WO2024022009A1 - 一种纳米蛋白降解工具、应用及其制备方法以及一种基于脂质的蛋白降解工具、应用及其制备方法 - Google Patents

一种纳米蛋白降解工具、应用及其制备方法以及一种基于脂质的蛋白降解工具、应用及其制备方法 Download PDF

Info

Publication number
WO2024022009A1
WO2024022009A1 PCT/CN2023/104199 CN2023104199W WO2024022009A1 WO 2024022009 A1 WO2024022009 A1 WO 2024022009A1 CN 2023104199 W CN2023104199 W CN 2023104199W WO 2024022009 A1 WO2024022009 A1 WO 2024022009A1
Authority
WO
WIPO (PCT)
Prior art keywords
lipid
degradation tool
protein
nanoparticles
poi
Prior art date
Application number
PCT/CN2023/104199
Other languages
English (en)
French (fr)
Inventor
师冰洋
郑蒙
刘洋
刘润涵
Original Assignee
河南大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN202210911853.5A external-priority patent/CN115260304A/zh
Priority claimed from CN202210906564.6A external-priority patent/CN115043932A/zh
Application filed by 河南大学 filed Critical 河南大学
Publication of WO2024022009A1 publication Critical patent/WO2024022009A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6893—Pre-targeting systems involving an antibody for targeting specific cells clearing therapy or enhanced clearance, i.e. using an antibody clearing agents in addition to T-A and D-M
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid

Definitions

  • the present disclosure belongs to the technical field of targeted drugs, and in particular relates to a nano-protein degradation tool, application and preparation method thereof, and a lipid-based protein degradation tool, application and preparation method thereof.
  • Targeted protein degradation tool specifically hijacks the protein of interest (POI, Protein of interest) to the intracellular protein recycling station to achieve targeted protein degradation.
  • POI protein of interest
  • proteolytic targeting chimeras As the most representative representative member of the first generation TPD, can target the degradation of traditionally difficult-to-drug targets, but the target Intracellular proteins only. Extracellular proteins and membrane proteins play an important role in the occurrence and development of diseases. About 40% of the total proteins encoded by genes are non-intracellular proteins.
  • LYTAC lysosome-targeting chimera
  • ASGPR asialoglycoprotein receptors
  • TPD tools require the design of 1 a suitable target protein recognition group, 2 a receptor-ligand matching pair to hijack the protein into cells and intracellular transport, 3 to initiate an appropriate protein degradation mechanism, and 4 from a drug-making perspective.
  • Targeting capabilities and biological barrier penetration capabilities are engineered specifically for cell types.
  • mainstream TPD tools including PROTAC, LYTAC and other similar tools, require laborious case design when developing tools for any new POI, many of which require de novo synthesis of compounds and a large number of screening processes before they can be used in different diseases and cell types. .
  • a nano-protein degradation tool including: one or more combinations of a first degradation tool, a second degradation tool, and a third degradation tool;
  • the first degradation tool is composed of a POI recognition group connected to a connecting arm;
  • the second degradation tool is composed of a POI recognition group connected to a nanoparticle;
  • the third degradation tool is composed of the POI recognition group The group is connected to the nanoparticle through the connecting arm;
  • the POI recognition group includes antibodies, proteins, polypeptides, nucleic acid aptamers or small molecules that can specifically bind to POI.
  • the POI recognition group and the connecting arm constitute a set of connecting units;
  • the first degradation tool is composed of multiple sets of the connecting arms located at the core and
  • the second degradation tool is composed of a connecting unit including the nanoparticles located at the core and a plurality of POI recognition groups connected to the nanoparticles and located on the periphery, with a multi-layer structure.
  • the nano-protein degradation tool is composed of a connecting unit including the nanoparticles located at the core and a plurality of POI recognition groups connected to the nanoparticles and located on the periphery, with a multi-layer structure.
  • the POI recognition group is connected to the nanoparticle through the connecting arm; the third degradation tool is composed of the nanoparticle located at the core, and the nanoparticle
  • the nano-protein degradation tool with a multi-layer structure is composed of multiple sets of connecting arms located in the middle layer and connecting units of the POI recognition groups located on the periphery and connected to the connecting arms. .
  • the connecting arm includes hydrophilic polymers, hydrophobic polymers and amphiphilic polymers.
  • the molecular weight of the connecting arm is 0-1000 kDa.
  • the amphiphilic polymer includes: amphiphilic block polymer, an amphiphilic polymer composed of the hydrophilic polymer and hydrophobic small molecules, and the hydrophobic polymer and hydrophilic small molecules. Amphiphilic polymer.
  • amphiphilic polymer is a polymer with a chain or branched molecular structure, which has at least one hydrophilic molecular end and one hydrophobic molecular end.
  • amphiphilic polymer has a linear molecular structure, with one end being a hydrophilic molecular end and the other end being a hydrophobic molecular end.
  • the nanoparticles include surface single nanoparticles and hybrid nanoparticles
  • the surface single nanoparticles include hydrophilic particles, hydrophobic particles and inorganic nanoparticles
  • the hybrid nanoparticles are hybrid nanoparticles modified by a hybrid substance on the surface of a single nanoparticle;
  • the hybrid substance is a modified membrane;
  • the modified membrane includes cell membranes, exosomes, oil films, hydrogels and liposomes.
  • the hybrid nanoparticles are particles composed of the modified film coating the outer surface of the surface single nanoparticle, so that the surface single nanoparticle is modified into the hybrid nanoparticle.
  • it can be connected to the arm of the hydrophilic polymer, the arm of the hydrophobic polymer, or the POI recognition group.
  • the particle size of the nanoparticles is 5-1000nm;
  • the nanoparticles are hydrophobic particles, hydrophilic particles or a single nanoparticle on the surface is coated with the modified particle.
  • the connecting arm is an amphiphilic polymer, a hydrophilic polymer or a hydrophobic polymer
  • the connecting arm is connected to the nanoparticle in a non-covalent manner.
  • the nanoparticles are covalently bonded to the nanoparticles or through active groups modified on the connecting arms.
  • the antibody in the POI recognition group is a therapeutic monoclonal antibody, a multispecific antibody, a nanobody, and the aforementioned antibody derivatives or antibody-conjugated drugs;
  • the polypeptide is a polypeptide with POI specific binding ability
  • the small molecule is a small molecule compound with POI specific binding ability.
  • the present disclosure also provides the application of the above-mentioned nano-protein degradation tool in the preparation of drugs, vaccines, and delivery vectors for treating and preventing abnormal protein accumulation diseases; wherein the abnormal protein accumulation diseases include tumors , immune system diseases, inflammation and pathogenic infections, neurodegenerative diseases, hematological diseases and metabolic diseases.
  • the present disclosure also provides a method for treating and preventing abnormal protein accumulation diseases, including administering a therapeutically effective amount of the above-mentioned nano-protein degradation tool to a subject, wherein the abnormal protein accumulation diseases include tumors , immune system diseases, inflammation and pathogenic infections, neurodegenerative diseases, hematological diseases and metabolic diseases.
  • the present disclosure also provides an application of the above-mentioned nano-protein degradation tool in preparing detection products and/or kits for abnormal protein accumulation diseases; wherein, the abnormal protein accumulation diseases include tumors, Immune system diseases, inflammation and pathogenic infections, neurodegenerative diseases, hematological diseases and metabolic diseases.
  • the abnormal protein accumulation diseases include tumors, Immune system diseases, inflammation and pathogenic infections, neurodegenerative diseases, hematological diseases and metabolic diseases.
  • the present disclosure also provides a method for preparing the above-mentioned nano-protein degradation tool, including:
  • the preparation of the first degradation tool and the second degradation tool includes: non-covalently bonding the POI recognition group to the nanoparticle or connecting arm, or alternatively, combining the POI recognition group based on The active group is covalently bonded to the nanoparticle or connecting arm to constitute the nanoprotein degradation tool;
  • the preparation of the third degradation tool includes:
  • the POI recognition group is coupled to the connecting arm to form a coupling intermediate; and then the coupling intermediate is connected to the core of the particle to form the third degradation tool; or
  • nanocomposite structure with connecting arms as the outer layer and nanoparticles as the core is constructed; then the POI recognition group is coupled to the composite structure of the nanoparticles to form the nanoprotein degradation tool.
  • the present disclosure provides a nano-protein degradation tool, application and preparation method thereof, wherein the nano-protein degradation tool includes: one or more combinations of a first degradation tool, a second degradation tool and a third degradation tool; wherein , the first degradation tool is composed of a POI recognition group connected to a connecting arm; the second degradation tool is composed of a POI recognition group connected to a nanoparticle; the third degradation tool is composed of the POI recognition group
  • the connecting arm is connected to the nanoparticle;
  • the POI recognition group includes an antibody, protein, polypeptide, nucleic acid aptamer or small molecule that can specifically bind to POI.
  • the present disclosure also provides a lipid-based protein degradation tool, including:
  • the POI recognition group includes antibodies, proteins, polypeptides, nucleic acid aptamers or small molecules that can specifically bind to POI;
  • the lipid hybrid substances include liposomes, exosomes, cell membranes and LNPs.
  • the lipid-based protein degradation tool is composed of the lipid hybrid substance in the core and the lipid hybrid substance in the periphery. Nanoparticles composed of the POI recognition group are used for protein degradation.
  • the lipid-based protein degradation tool further includes the lipid-based protein degradation tool provided with a connecting member between the POI recognition group and the lipid hybrid substance.
  • the molecular weight of the connecting member is 0-1000kDa;
  • the connecting member is one of a polymer connecting arm and a lipid connecting arm.
  • the polymer linker includes hydrophilic polymers, hydrophobic polymers and amphiphilic polymers.
  • the lipid linking arm is an amphipathic lipid linking arm.
  • the POI recognition group and the connection member constitute a set of connection units;
  • the lipid-based The protein degradation tool is composed of the lipid hybrid material located at the core, a plurality of sets of connecting members located in the middle layer and connected to the lipid hybrid material, and a set of connecting members located at the periphery and connected to the lipid hybrid material.
  • the lipid-based protein degradation tool with a multi-layer structure is composed of multiple sets of connecting units of the POI recognition group connected to the connecting member.
  • the lipid connecting arm includes at least two ends, one end is a lipophilic end that can be connected to the lipid hybrid substance, and the other end is a hydrophilic end.
  • the lipophilic end is a lipid molecule.
  • amphiphilic polymer is a polymer with a chain or branched molecular structure, which has at least one hydrophilic molecular end and one hydrophobic molecular end.
  • amphiphilic polymer has a linear molecular structure, with one end being a hydrophilic molecular end and the other end being a hydrophobic molecular end.
  • nanoparticles are also included, wherein the nanoparticles are coated with the lipid hybrid substance at the core of the lipid-based protein degradation tool;
  • the nanoparticles include hydrophilic particles, hydrophobic particles and inorganic nanoparticles.
  • the particle size of the nanoparticles is 5-1000 nm.
  • the present disclosure also provides a lipid-based protein degradation tool as mentioned above, for use in preparing drugs and delivery systems for treating and preventing abnormal accumulation of harmful proteins; wherein, the harmful protein is abnormal Accumulating diseases include tumors, immune system diseases, neurodegenerative diseases, hematological diseases and metabolic diseases.
  • the present disclosure also provides a method for treating and preventing abnormal protein accumulation diseases, including administering a therapeutically effective amount of the above-mentioned lipid-based protein degradation tool to a subject, wherein the abnormal protein accumulation Diseases include tumors, immune system diseases, inflammation and pathogenic infections, neurodegenerative diseases, hematological diseases and metabolic diseases.
  • the present disclosure also provides a lipid-based protein degradation tool as mentioned above, which can be used to prepare diseases related to abnormal accumulation of harmful proteins.
  • the diseases with abnormal accumulation of harmful proteins include tumors, immune system diseases, neurodegenerative diseases, blood system diseases and metabolic diseases.
  • the present disclosure also provides a method for preparing the above-mentioned lipid-based protein degradation tool, where the lipid-based protein degradation tool is the POI recognition group and the lipid hybrid.
  • the preparation method is: non-covalently bonding the POI recognition group to the lipid hybrid substance; or, coupling the POI recognition group to the lipid hybrid substance The group is covalently bonded to the lipid hybrid substance to constitute the lipid-based protein degradation tool.
  • the lipid-based protein degradation tool further includes a protein degradation tool in which the POI recognition group is connected to the lipid hybrid substance through a connecting member;
  • the preparation method is:
  • the POI recognition group is coupled to the connecting member to form a coupling intermediate; then the coupling intermediate is connected to the lipid hybrid substance to form the lipid-based protein degradation Tools; or
  • the present disclosure provides a lipid-based protein degradation tool, application and preparation method, wherein the lipid-based protein degradation tool includes: a POI recognition group, and a lipid complex connected to the POI recognition group. hybrid substances; wherein, the POI recognition group includes antibodies, proteins, polypeptides, nucleic acid aptamers or small molecules that can specifically bind to POI; the lipid hybrid substances include liposomes, exosomes, cell membranes and LNP.
  • Figure 1 is a schematic structural diagram of the first degradation tool of the nano-protein degradation tool of the present disclosure
  • Figure 2 is a schematic structural diagram of the second degradation tool of the nano-protein degradation tool of the present disclosure
  • Figure 3 is a schematic structural diagram of the third degradation tool of the nano-protein degradation tool of the present disclosure.
  • Figure 4 is a schematic diagram of the synthesis of the first degradation tool in Example 1 of the present disclosure
  • Figure 5 is the Coomassie Brilliant Blue staining result of the first degradation tool in Example 1 of the present disclosure
  • Figure 6 is a protein electrophoresis result of the protein degradation effect of the first degradation tool in Example 1 of the present disclosure
  • Figure 7 is a schematic diagram of the synthesis of the second degradation tool in Example 2 of the present disclosure.
  • Figure 8 shows the JQ1-NH 2 structure used in the second degradation tool JQ1-NP nanoparticles in Example 2 of the present disclosure and the dynamic light scattering results of JQ1-NP nanoparticles;
  • Figure 9 is the electrophoresis result of the second degradation tool JQ1-NP protein in Example 2 of the present disclosure.
  • Figure 10 is the electrophoresis result of the second degradation tool NTZ-NP2 protein in Example 2 of the present disclosure
  • Figure 11 is a schematic diagram of the synthesis of the third degradation tool in Example 3 of the present disclosure.
  • Figure 12 is a schematic structural diagram of the third degradation tool in Embodiment 3 of the present disclosure.
  • Figure 13 is the dynamic light scattering and transmission electron microscopy results of the third degradation tool in Example 3 of the present disclosure.
  • Figure 14 shows the protein electrophoresis results of the third degradation tool (NTZ-NP) in Example 3 of the present disclosure on the protein degradation effect of human breast cancer M231 cells, human cervical cancer HeLa cells, and human brain glioma U87 cells;
  • Figure 15 shows the immunofluorescence staining results of M231 cells after being treated with the third degradation tool
  • Figure 16 shows the degradation results of green fluorescently labeled EGFR protein taken in a fixed field of view live cell time series of M231 cells after being treated with the third degradation tool
  • Figure 17 shows the cell viability results of CCK8 detection of HepG2 cells after being treated with the third degradation tool
  • Figure 18 shows the EGFR protein expression of the third degradation tool in Example 3 of the present disclosure; where a is the exploration of effective degradation concentration; b is the exploration of protein recovery after treatment;
  • Figure 19 shows the expression of EGFR protein of the third degradation tool in Example 3 of the present disclosure, exploring the different proportions between the coupling intermediate composed of a POI recognition group and a connecting arm and a blank connecting arm without a POI recognition group. The impact of degradation effects;
  • Figure 20 is a schematic structural diagram of the third degradation tool (NTZ-AuNP) in Example 4 of the present disclosure.
  • Figure 21 is the electrophoresis result of the protein degradation effect of the third degradation tool (NTZ-AuNP) in Example 4 of the present disclosure
  • Figure 22 is the hydrogen nuclear magnetic resonance spectrum ( 1 H NMR) of the POI recognition group and connecting arm connection product (ACE2-PEG-DSPE) in the third degradation tool (ACE2-NP) in Example 5 of the present disclosure;
  • Figure 23 is the hydrogen nuclear magnetic resonance spectrum ( 1 H NMR) of the product (CD13-PEG-DSPE) connecting the POI recognition group and the connecting arm in the third degradation tool (CD13-NP) in Example 5 of the present disclosure;
  • Figure 24 is a diagram showing the results of flow cytometry detection of GFP after the third degradation tool (ACE2-NP) treated 293 cells labeled with GFP ACE2 in Example 5 of the present disclosure;
  • Figure 25 is a picture of the protein electrophoresis results after the third degradation tool (CD13-NP) treated human liver cancer cell HepG2 in Example 5 of the present disclosure
  • Figure 26 shows the experimental results of confocal microscopy detecting the endocytosis of FITC green dye-labeled ⁇ -amyloid 1-42 oligomers after treating human glial cells with the third degradation tool (AB-NP) in Example 5 of the present disclosure. ;
  • Figure 27 is the hydrogen nuclear magnetic resonance spectrum ( 1 H NMR) of the POI recognition group and connecting arm connection product (Palb-PEG-DSPE) in the third degradation tool (Palb-NP) in Example 6 of the present disclosure;
  • Figure 28 is a protein electrophoresis diagram of the protein degradation effect of the third degradation tool (Palb-NP) in Example 6 of the present disclosure.
  • Figure 29 is the result of crystal violet staining colony formation experiment after M231 cells were treated with the third degradation tool in Example 6 of the present disclosure.
  • Figure 30 is a schematic structural diagram of the third degradation tool (AV45-NP) in Example 6 of the present disclosure.
  • Figure 31 is the NMR spectrum of the POI recognition group and connecting arm connection product (AV45-PEG-DSPE) in the third degradation tool (AV45-NP) in Example 6 of the present disclosure. vibration hydrogen spectrum;
  • Figure 32 is the experimental results of confocal microscopy detecting the endocytosis of FITC green dye-labeled ⁇ -amyloid 1-42 oligomers after treating human glial cells with the third degradation tool (AV45-NP) in Example 6 of the present disclosure. ;
  • Figure 33 is a protein electrophoresis diagram of the protein degradation effect of the third degradation tool (CTX-NP) in Example 7 of the present disclosure
  • Figure 34 is a confocal microscope image of the protein degradation effect of the third degradation tool (PTZ-NP) in Example 7 of the present disclosure
  • Figure 35 is a protein electrophoresis diagram of the protein degradation effect of the third degradation tool (ATZ-NP) in Example 7 of the present disclosure.
  • Figure 36 is a protein electrophoresis diagram of the protein degradation effect of the third degradation tool (CRLZ-NP) in Example 7 of the present disclosure
  • Figure 37 is a protein electrophoresis diagram of the protein degradation effect of the third degradation tool (INE/NTZ-NP) in Example 7 of the present disclosure.
  • Figures 38 and 39 are animal in vivo experiments of the third degradation tool (NTZ-NP) in Example 8 of the present disclosure; wherein, a in Figure 38 is the nude mouse tumor volume during the treatment of NTZ-NP in the M231 cell nude mouse subcutaneous tumor model; b: After NTZ-NP treatment, tumors were dissected and separated for protein electrophoresis to detect EGFR expression and statistics; c: Body weight changes of nude mice during NTZ-NP treatment;
  • Figure 39 shows the results of immunofluorescence staining to detect EGFR after the tumor tissue was separated from the NTZ-NP treatment
  • Figure 40 shows the results of separating tumor tissue after NTZ-NP treatment and performing immunofluorescence staining to detect apoptosis markers
  • Figure 41 shows the detection of blood, liver and kidney function indicators in white mice after a single administration of the third degradation tool (NTZ-NP) in Example 8 of the present disclosure
  • Figure 42 shows that the third degradation tool (NTZ-NP) in Example 8 of the present disclosure obtains blood-brain barrier crossing ability and tumor targeting ability through simple self-assembly, and is loaded with fluorescent dye DIR for tracking, and is traced through a small animal imager Detect brain aggregation and statistical graphs of DIR fluorescence;
  • Figure 43 is a diagram showing the results of immunohistochemical detection of EGFR and proliferation marker PCNA after brain tumor dissection after the third degradation tool (NTZ-NP) in Example 8 of the present disclosure was administered to the glioma orthotopic animal model through the assembly method of Figure 42;
  • Figure 44 is a schematic structural diagram of the coupling of the POI recognition group and the lipid hybrid substance of the present disclosure
  • Figure 45 is a schematic structural diagram of the coupling of a POI recognition group containing a connecting component and a lipid hybrid substance according to the present disclosure
  • Figure 46 is a schematic structural diagram of the connection between a POI recognition group containing a connecting component-lipid linking arm and a lipid hybrid substance according to the present disclosure
  • Figure 47 is a schematic diagram of the synthesis structure of NTZ-PEGlipo in Example 9 of the present disclosure.
  • Figure 48 is the protein electrophoresis result of the degradation effect of NTZ-PEGlipo on the target protein EGFR in M231 cells in Example 9 of the present disclosure
  • Figure 49 is a schematic diagram of the synthesis of NTZ-lipo1, NTZ-lipo2, INE-lipo, Palb-lipo, and AV45-lipo in Example 10 of the present disclosure;
  • Figure 50 shows the protein electrophoresis results of different concentrations of NTZ-lipo1 on the degradation of the target protein EGFR in M231 cells in Example 10 of the present disclosure
  • Figure 51 shows the protein electrophoresis results of the degradation effect of NTZ-lipo1 with different lipid hybrid substance composition ratios on the target protein EGFR in M231 cells in Example 10 of the present disclosure
  • Figure 52 is the protein electrophoresis result of the degradation effect of NTZ-lipo2 on the target protein EGFR in M231 cells in Example 10 of the present disclosure
  • Figure 53 is the protein electrophoresis results of the degradation effect of INE-lipo on the target protein HER2 in M231 cells in Example 10 of the present disclosure
  • Figure 54 is the hydrogen nuclear magnetic resonance spectrum ( 1 H NMR) result of the product after coupling the lipid connecting arm used in Palb-lipo with the POI recognition group in Example 10 of the present disclosure;
  • Figure 55 is the proton nuclear magnetic resonance spectrum ( 1 H NMR) result of the product after coupling the lipid connecting arm used in AV45-lipo with the POI recognition group in Example 10 of the present disclosure;
  • Figure 56 shows the protein electrophoresis results of different concentrations of Palb-lipo on the degradation of the target protein CDK4 in M231 cells in Example 10 of the present disclosure
  • Figure 57 is a schematic structural diagram of AV45-lipo in Embodiment 10 of the present disclosure.
  • Figure 58 shows the co-incubation of AV45-lipo and ⁇ -amyloid (A ⁇ ) oligomers in human glial cell culture medium in Example 10 of the present disclosure, followed by confocal microscopy detection of cell lysosomal markers and fluorescent dye FITC after washing with PBS. Results of autofluorescence of labeled A ⁇ and AV45;
  • Figure 59 is a schematic structural diagram of NTZ-LNP1, NTZ-LNP1s, and NTZ-LNP2 in Example 11 of the present disclosure
  • Figure 60 is the dynamic light scattering (DLS) particle size distribution chart results of NTZ-LNP1, NTZ-LNP1s, and NTZ-LNP2 in Example 11 of the present disclosure;
  • Figure 61 is the protein electrophoresis results of the degradation effect of NTZ-LNP1 in Example 11 of the present disclosure and NTZ-lipo1 in Example 10 on the target protein EGFR in M231 cells;
  • Figure 62 is the protein electrophoresis result of the degradation effect of NTZ-LNP1s on the target protein EGFR in M231 cells in Example 11 of the present disclosure
  • Figure 63 is the protein electrophoresis result of the degradation effect of NTZ-LNP2 on the target protein EGFR in M231 cells in Example 11 of the present disclosure
  • Figure 64 is a DLS particle size distribution and average dispersion coefficient PDI diagram of NTZ-exo in Example 12 of the present disclosure
  • Figure 65 is the protein electrophoresis result of the degradation effect of NTZ-exo on the target protein EGFR in M231 cells in Example 12 of the present disclosure
  • Figure 66 is a schematic structural diagram of NTZ-lipoP in Example 13 of the present disclosure.
  • Figure 67 shows the protein electrophoresis results of different concentrations of NTZ-lipoP on the degradation of the target protein EGFR in M231 cells in Example 13 of the present disclosure
  • Figure 68 is a schematic structural diagram of CTX-RBCmD in Example 13 of the present disclosure.
  • Figure 69 is a DLS particle size distribution result diagram of CTX-RBCmD in Example 13 of the present disclosure.
  • Figure 70 shows the protein electrophoresis results of the degradation effect of CTX-RBCmD on the target protein EGFR in M231 cells in Example 13 of the present disclosure.
  • Lipid-based protein degradation tools 1. POI recognition group; 2. Liposomes, exosomes or cell membranes in lipid hybrid substances; 3. LNP in lipid hybrid substances; 4. Lipid hybrid substances 5, polymer connecting arm; 6, coupling group; 7, nanoparticles.
  • the terms “includes,” “includes,” “has,” “contains,” or “involves” are inclusive or open-ended and do not exclude others. Unlisted elements or method steps.
  • the term “consisting of” is considered an alternative embodiment of the term “comprising”. If in the following a certain group is defined as containing at least a certain number of embodiments, this is also to be understood as revealing a group that optionally consists only of these embodiments.
  • the present disclosure provides a nano-protein degradation tool, including:
  • One or more combinations of the first degradation tool for its structure, refer to Figure 1), the second degradation tool (for its structure, refer to Figure 2), and the third degradation tool (for its structure, refer to Figure 3);
  • the first degradation tool is composed of a POI recognition group connected to a connecting arm;
  • the second degradation tool is composed of a POI recognition group connected to a nanoparticle;
  • the third degradation tool is composed of the POI recognition group The group is connected to the nanoparticle through the connecting arm;
  • the POI recognition group includes antibodies, proteins, polypeptides, nucleic acid aptamers or small molecules that can specifically bind to POI.
  • the POI recognition group is an antibody, protein, polypeptide, nucleic acid aptamer or small molecule that can specifically bind to POI.
  • the nanoparticles are nanoparticles or nanoparticles, which can be a solid particle structure, a hollow structure, or a porous particle structure.
  • the component or molecular structure in the particle structure can be a single component or a multi-component composite.
  • the nanoparticles may be biocompatible nanoparticles.
  • the connecting arm is a structure and substance that can help improve water solubility and stability, and serves to connect with the POI recognition group, or to serve as a bridge between the POI recognition group and the nanoparticles to form an overall structure. .
  • the nano-protein degradation tool includes three expression forms (structural forms), which are the first degradation tool, the second degradation tool and the third degradation tool.
  • the structural form is shown in the following table:
  • TPD-NP nano-protein degradation tools
  • the POI recognition group includes: antibodies, proteins, polypeptides, nucleic acid aptamers or small molecules that can specifically bind to POI.
  • antibodies can include therapeutic monoclonal antibodies, multispecific antibodies, nanobodies, and the aforementioned antibody derivatives or antibody conjugates;
  • the protein can be: a protein with POI specific binding ability
  • Polypeptides are polypeptides with specific binding ability to POI; including binding peptides of ACE2, CD13, and ⁇ -Amyloid;
  • Nucleic acid aptamers are nucleic acid aptamers with POI-specific binding ability
  • Small molecules are small molecule compounds with POI-specific binding ability, including derivatives of the CDK4/6 protein inhibitor Palbociclib, derivatives of the BRD4 protein inhibitor JQ1, and ⁇ -Amyloid protein probe AV-45 Derivatives of Pittsburgh compound PiB, derivatives of Tau protein probes GTP1 and PBB3.
  • the antibodies include: CTX, NTZ, PTZ, CRLZ, INE, ATZ, AND and MTX.
  • CTX (Cetuximab) is the most classic human-mouse chimeric EGFR protein monoclonal antibody.
  • NTZ Non-timotuzumab
  • PTZ pertuzumab
  • HER2-targeting monoclonal antibody drug developed and marketed by Roche in 2012. It has a different binding site from trastuzumab to the HER2 protein.
  • CRLZ (Camrelizumab) PD-1 inhibitor
  • Camrelizumab is a classic immunotherapy monoclonal antibody used for the treatment of advanced lung cancer, liver cancer, esophageal cancer and Hodgkin lymphoma.
  • INE Inetetamab
  • ATZ (Atezolizumab) is a humanized monoclonal antibody against PD-L1 and a classic tumor immunotherapy target.
  • Aducanumab ADN (aducanumab) is a monoclonal antibody used for Alzheimer's disease that binds to ⁇ -amyloid protein.
  • MTX (Miltuximab) is a monoclonal antibody against glypican 1 and an emerging tumor treatment target.
  • the POI recognition group is modified on the surface of the nanoparticle, or the POI recognition group is connected to the connecting arm, or the POI recognition group is modified on the connecting arm and connected to the surface of the nanoparticle through the connecting arm, and the POI recognition group is exposed after assembly.
  • nanoparticles can penetrate cells without relying on specially designed receptor-ligand matching pairs, and can be coupled to POI recognition groups such as small molecules, peptides, nucleic acid aptamers, and antibodies. , specifically perform protein hijacking and targeted degradation, thereby achieving the assembly of TPD tools (TPD-NP) based on nanoparticles (NP, Nanoparticles).
  • TPD-NP nanoprotein degradation tool with simple drug loading and tissue specificity makes drug and protein degradation tool therapy and translational/precision medicine possible.
  • the invention of TPD-NP and the exploration of its mechanism have greatly expanded the scope of TPD tools and provided basic knowledge for the fields of TPD and nanodelivery, and can in principle degrade a variety of human disease-related extracellular/membrane-associated/ intracellular proteins.
  • the overall three degradation tools have a spatial structure as shown in the following table after construction:
  • TPD-NP nano-protein degradation tools
  • the nano-protein degradation tool has different spatial structures of TPD-NP due to different connection methods.
  • the POI recognition group and the connecting arm form a set of connection units, the whole is a multi-layer structure, with the core inside and the periphery outside. Multiple connection units are placed into the multi-layer structure.
  • its form is that one end of the connecting arm is at the core, and the POI recognition group connected to the connecting arm is on the periphery, forming a granular TPD-NP with a multi-layered structure from the inside to the outside.
  • the POI recognition group is connected to the nanoparticles, its spatial structure is also composed of a multi-layer structure, with the inner core and the outer periphery, and the inner core is a single nanoparticle.
  • the multiple POI recognition groups connected to it are on the periphery, forming a second degradation tool in the form of particles with an inner and outer double-layer structure.
  • the third degradation tool because its connection method is that the POI recognition group is connected to the nanoparticle through the connecting arm, that is, "POI recognition group-connecting arm-nanoparticle".
  • the overall structure is multi-layered, divided into three layers, with the inner part being the core, the middle part being the middle layer, and the outer part being the periphery.
  • the internal core is a nanoparticle, and multiple connecting arms connected to the nanoparticle are in the middle layer.
  • there are POI recognition groups connected to these connecting arms forming a three-layer structure of particle form.
  • the linking arms include hydrophilic polymers, hydrophobic polymers and amphiphilic polymers;
  • the connecting arm can be divided into three different connecting arms, including: 1. Hydrophilic polymer, 2. Hydrophobic polymer, 3. Amphiphilic polymer.
  • the hydrophilic polymer may include, but is not limited to: polyethylene glycol (PEG), polyethylene oxide (PEO), poly(ethylene glycol) methacrylate (POEG), poly(2-methacryloyl) Oxyethyl phosphoryl (PMPC), polycarboxylic acid betaine (PCB), dextran, hyaluronic acid, chitosan, ⁇ -cyclodextrin, hyperbranched polyglycidyl ether (HPG), polyN- (2-Hydroxypropyl)methacrylamide (PHPMA), polyhydroxyethyl methacrylate (PHEMA), polyacrylamide (PAM), polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), polymaleic anhydride (HPMA), polyquaternary ammonium salts and pharmaceutically acceptable polymer salts thereof, polyethylenimine (PEI), poly N,N-dimethylaminoethyl methacrylate (PDMAEMA), polylysine
  • the hydrophobic polymer may include, but is not limited to: polylactic acid-co-glycolic acid (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polycarbonate (PMC) and its derivatives, glycolide /Copolymers of various combinations and components of lactide/caprolactone/carbonate, polyurethane (PU), polyether ether ketone (PEEK), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA) , polyethylene (PE), polyphenylalanine and other types of hydrophobic polyamino acids and derivatives of the above polymers and their pharmaceutically acceptable salts.
  • PLGA polylactic acid-co-glycolic acid
  • PLA polylactic acid
  • PCL polycaprolactone
  • PMC polycarbonate
  • glycolide /Copolymers of various combinations and components of lactide/caprolactone/carbonate
  • PU polyurethane
  • PEEK polyether ether ketone
  • PMMA
  • the amphiphilic polymer may include, but is not limited to: PEG-PLGA, PEG-PCL, PEG-PLA, PEG-PMC, and various amphiphilic block polymers that are combinations of the above-mentioned hydrophilic polymers and hydrophobic polymers. and derivatives, as well as various amphiphilic polymers and derivatives composed of the above-mentioned hydrophilic polymers and hydrophobic molecules (such as lipids).
  • lipid molecules may include, but are not limited to: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, pregnenols Prenol lipids, saccharolipids, polyketides, cationic lipids and ionizable lipids and their derivatives.
  • the molecular weight of the connecting arm is 0-1000kDa;
  • the amphiphilic polymer includes: an amphiphilic block polymer, an amphiphilic polymer composed of the hydrophilic polymer and a hydrophobic small molecule, and an amphiphilic polymer composed of the hydrophobic polymer and a hydrophilic small molecule. Amphiphilic polymer.
  • amphiphilic polymers include three types, and their structural forms are shown in the following table:
  • amphiphilic block polymers refer to linear or branched ones. There are two or more structurally different segments in a single polymer molecule. Copolymers with specific chemical structures and molecular weights can be synthesized as needed.
  • Amphiphilic block polymers refer to self-assembly into specific supramolecular ordered aggregates (micelles or vesicles) in solution.
  • Amphiphilic block polymers refer to polymer micelles composed of a hydrophilic shell and a lipophilic core, or polymer vesicles composed of a hydrophilic shell, a lipophilic middle layer and a hydrophilic cavity, which can spontaneously form after being dissolved in water.
  • amphiphilic polymers composed of hydrophilic polymers and hydrophobic small molecules
  • amphiphilic polymers composed of hydrophobic polymers and hydrophilic small molecules.
  • the amphiphilic polymer is a polymer with a chain or branched molecular structure, which has at least one hydrophilic molecular end and one hydrophobic molecular end.
  • the amphiphilic polymer is a polymer with a chain molecular structure, which can be a polymer with a molecular structure composed of multiple straight chains connected, and its molecular structure has at least one hydrophilic molecular end and one hydrophobic molecule. end, thus making the amphiphilic polymer amphiphilic.
  • amphiphilic polymer has a linear molecular structure, with one end being a hydrophilic molecular end and the other end being a hydrophobic molecular end.
  • the amphiphilic polymer has a linear structure, and the linear molecular structure includes two molecular ends, one of which is a hydrophilic molecular end and the other end is a hydrophobic molecular end.
  • the function of the connecting arm can be to modify the nanoparticles with water solubility or improve the steric hindrance of the POI recognition group. Therefore, when it is necessary to increase the water solubility of nanoparticles and improve the steric hindrance of the POI recognition group, it is necessary to use connecting arms to connect the nanoparticles.
  • nanoparticles are fat-soluble polymers, water-soluble polymers or inorganic nanoparticles in organic polymer nanoparticles, it is necessary to use connecting arms in the nanoprotein degradation tool to improve the stability of the nanoparticles and bridge the nanoparticles and POI between recognition groups.
  • the nanoparticle When the nanoparticle is an amphiphilic polymer, it has both hydrophilic and hydrophobic amphiphilic properties, so there is no need to increase or improve its hydrophilicity. At this time, there is no need to use the function of the connecting arm and directly use the nanoparticles.
  • the particles are linked to POI recognition groups.
  • the nanoparticles include surface single nanoparticles and hybrid nanoparticles
  • the surface single nanoparticles include hydrophilic particles, hydrophobic particles and inorganic nanoparticles.
  • the inorganic nanoparticles include 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 Particles, aluminum-iron alloy particles, and titanium-iron alloy particles are all materials with stable and low biological toxicity.
  • the single nanoparticle can be a biocompatible material.
  • the single nanoparticle is a biodegradable material.
  • the hydrophilic particles may be dendritic polymers, hyperbranched polymers, various nanogels composed of the aforementioned hydrophilic polymers and their derivatives, and nanoalbumin.
  • the hydrophobic particles can be polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polycarbonate (PMC) and its derivatives, glycolide/lactide /Caprolactone/carbonate copolymer, polyurethane (PU), polyether ether ketone (PEEK), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), polystyrene (PS), polyethylene Nanoparticles prepared from various hydrophobic polyamino acids and their derivatives such as (PE) and polyphenylalanine.
  • the hybrid nanoparticles are hybrid nanoparticles modified by a hybrid substance on the surface of a single nanoparticle.
  • the hybrid substance is a modified membrane;
  • the modified membrane includes cell membranes, exosomes, oil films, hydrogels and liposomes.
  • the hybrid substance may be a modified membrane.
  • the nanoparticles are coated with a modified membrane so that they have a certain change in water solubility.
  • the modified membrane may include cell membranes, exosomes, oil films, hydrogels and liposomes.
  • the hybrid nanoparticles are particles formed by coating the outer surface of the surface single nanoparticle with the modified film, so that the surface single nanoparticle is modified into the hybrid nanoparticle.
  • it can be connected to the arm of the hydrophilic polymer or the hydrophobic polymer, or to the arm of the amphiphilic polymer, or to the POI recognition group.
  • the particle size of the nanoparticles is 5-1000nm;
  • the nanoparticles are hydrophobic particles, hydrophilic particles or hybrid nanoparticles in which the single nanoparticle on the surface is coated with the modified film.
  • the connecting arm is an amphiphilic polymer, a hydrophilic polymer or a hydrophobic polymer
  • the connection method between the connecting arm and the nanoparticle includes: the connecting arm is non-covalently bonded to the nanoparticle. on the particle, or covalently bonded to the nanoparticle through an active group modified on the connecting arm;
  • the antibody in the POI recognition group is a therapeutic monoclonal antibody, a multispecific antibody, a nanobody, and the aforementioned antibody derivatives or antibody-conjugated drugs;
  • the polypeptide is a polypeptide with POI specific binding ability; it includes binding polypeptides of ACE2, CD13, and ⁇ -Amyloid;
  • Nucleic acid aptamers are nucleic acid aptamers with POI-specific binding ability
  • the small molecules are small molecule compounds with POI specific binding ability, including derivatives of the CDK4/6 protein inhibitor Palbociclib, derivatives of the BRD4 protein inhibitor JQ1, and ⁇ -Amyloid protein probe AV Derivatives of -45, derivatives of Pittsburgh compound PiB, derivatives of Tau protein probes GTP1 and PBB3.
  • CTX is the most classic human-mouse chimeric EGFR protein monoclonal antibody. It has been approved by more than 100 countries/regions around the world for the treatment of RAS wild-type metastatic colorectal cancer, locally advanced and Recurrent and metastatic head and neck squamous cell carcinoma.
  • NTZ Nemotuzumab
  • NTZ is the most classic EGFR protein monoclonal antibody in China and is used to treat a variety of tumors caused by EGFR mutations.
  • PTZ Pertuzumab
  • CRLZ Camrelizumab
  • Camrelizumab is a classic immunotherapy monoclonal antibody used for the treatment of advanced lung cancer, liver cancer, esophageal cancer and Hodgkin lymphoma.
  • INE Inetetamab
  • ATZ is a humanized monoclonal antibody against PD-L1 and a classic tumor immunotherapy target.
  • Aducanumab ADN is a monoclonal antibody used for Alzheimer's disease that binds to ⁇ -amyloid protein.
  • MTX Miltuximab
  • glypican 1 is an emerging tumor treatment target.
  • the present disclosure also provides a nano-protein degradation tool for use in the preparation of drugs, vaccines, and delivery vectors for the treatment and prevention of abnormal protein accumulation diseases; wherein the abnormal protein accumulation diseases include tumors, immune system diseases, and neurological diseases. Degenerative diseases, hematological diseases and metabolic diseases.
  • the present disclosure also provides a nano-protein degradation tool for use in preparing detection products and/or kits for abnormal protein accumulation diseases; wherein the abnormal protein accumulation diseases include tumors, immune system diseases, inflammation and Pathogen infections, neurodegenerative diseases, hematological diseases and metabolic diseases.
  • abnormal protein accumulation diseases include tumors, immune system diseases, inflammation and Pathogen infections, neurodegenerative diseases, hematological diseases and metabolic diseases.
  • such protein degradation tools are used to degrade a variety of pathologically relevant proteins, and in situ tumor pathology-related protein degradation is experimentally performed in animal tumor models.
  • Applications include but are not limited to drug development and diagnosis in diseases involving abnormal accumulation of harmful proteins, such as tumors, immune system diseases, inflammation and pathogenic infections, neurodegenerative diseases, hematological diseases and metabolism-related diseases, as well as in vitro testing and biomedicine Development of protein degradation tools in research, and kit development in protein interaction studies.
  • the present disclosure also provides a method for preparing a nano-protein degradation tool, which is characterized by including:
  • Preparation method 1 for the first or second degradation tool non-covalently bond the POI recognition group to the nanoparticle or connecting arm;
  • Preparation method 2 for the first or second degradation tool covalently bond the POI recognition group to the nanoparticle or connecting arm based on the active group, thereby forming the nanoparticle Protein degradation tools;
  • Preparation method 1 for the third degradation tool first couple the POI recognition group to the connecting arm to form a coupling intermediate; then connect the coupling intermediate to the The core part of the particle constitutes the third degradation tool;
  • Preparation method 2 for the third degradation tool first construct a nanocomposite structure with connecting arms as the outer layer and nanoparticles as the core; then combine the POI recognition group with the nanoparticles Structural coupling constitutes the nano-protein degradation tool.
  • Degradation tool Select the corresponding method to prepare the corresponding protein degradation tool.
  • (1) and (2) are two methods for preparing the first or second degradation tool respectively, which can be selected according to the properties of the nanoparticles
  • (3) and (4) are two methods for preparing the third degradation tool. The difference between the methods is that the connection sequence is different.
  • the POI recognition group is first coupled to the connecting arm and then connected to the core of the particle.
  • the connecting arm is first connected to the nanoparticle to form After the composite structure is coupled with the POI recognition group.
  • the present disclosure also provides a lipid-based protein degradation tool, including:
  • the POI recognition group includes antibodies, proteins, polypeptides, nucleic acid aptamers or small molecules that can specifically bind to POI;
  • the lipid hybrid substances include liposomes, exosomes, cell membranes and LNP (lipid nanoparticle).
  • the POI recognition group is an antibody, protein, polypeptide or small molecule that can specifically bind to POI.
  • the lipid hybrid substances include liposomes, exosomes, cell membranes and LNPs.
  • LNP is lipid nanoparticle.
  • lipid nanoparticles are mainly used for in vivo drug delivery. From traditional liposomes to lipid nanoparticles (LNP, lipid nanoparticles), they have been used in small molecule drug delivery, nucleic acid drug delivery, Nano-vaccines and other fields are widely used in medical treatment. Among them, most of the COVID-19 mRNA vaccines are composed of LNP.
  • Lipid nanoparticles have the advantage of being low-cost and easy to prepare, and their composition rules have also been studied clearly. Lipid nanoparticles used for medical treatment and drug development have a relatively regular composition, the most basic of which are membrane skeleton and cholesterol. Excipients. Lipid nanoparticles in clinical use can also be added with other components as needed, which can include but are not limited to the following components:
  • Membrane skeleton the main component of the lipid bilayer membrane.
  • auxiliary phospholipids which maintain the microscopic morphology of liposomes and destabilize lysosomal membranes.
  • PEGylated lipids reduce the binding of particles to plasma proteins in the body and prolong systemic circulation time.
  • Cationic lipids highly efficient in encapsulating nucleic acid drugs, providing positive charge, transfection in vivo, pH sensitive (ionizable).
  • Stabilizer which has a freeze-drying protective effect and maintains the structural stability of liposomes during the freeze-drying process.
  • the lipid hybrid substances include liposomes, exosomes, cell membranes, LNP, LPP and lipid nanoemulsions.
  • liposomes also known as classic liposomes, have a vacuole shape and are mainly used to carry hydrophobic drugs. Since liposomes are an amphiphilic membrane, hydrophobic vacuoles can carry hydrophobic drugs, and hydrophilic vacuoles can carry hydrophilic drugs.
  • LNP is a lipid nanoparticle that contains cationic lipids inside.
  • mRNA vaccines and some nucleic acid vaccines use LNP. Because nucleic acids are negatively charged and cationic lipids are positively charged, they can effectively improve the encapsulation efficiency.
  • LNP is also Used for the delivery of CRISPR gene editing elements.
  • traditional liposomes have low efficiency in loading nucleic acids and generally carry hydrophobic and hydrophilic small molecule drugs.
  • LPP contains a polymer core, it obtains the separate properties of lipids and polymers, and has better drug loading compatibility and stability.
  • LNP and LPP also have the advantage of being easily modified and transformed.
  • Liposomes and LNPs can be easily modified through amphipathic PEGylated lipids and similar structures.
  • PEGylated lipids can be formulated through PEG end-linking.
  • Ligands are used to bind to receptors and may promote drug delivery to target organs. They are also known as active-targeting lipid nanocarriers.
  • Lipid hybrid substances facilitate drug loading, and the drugs include hydrophilic and hydrophobic small molecule drugs, nucleic acid drugs, protein drugs, gene editing vectors and other biotech drugs that can be effectively loaded and delivered.
  • the drugs include hydrophilic and hydrophobic small molecule drugs, nucleic acid drugs, protein drugs, gene editing vectors and other biotech drugs that can be effectively loaded and delivered.
  • its application is still focused on drug delivery. The physiological and pathological properties of its structure still need to be further explored, and more therapeutic potential and possibilities are worthy of exploration.
  • the present disclosure achieves target protein degradation based on lipid hybrid substances by modifying the POI recognition group on the surface of the lipid hybrid substance, and exposing the POI recognition group to the outside of the lipid hybrid substance after assembly. , which greatly reduces the difficulty of synthesizing protein degradation tools.
  • a large number of compounds, peptides, antibodies and nucleic acid aptamers that have binding ability to target proteins can be upgraded into protein degradation drugs. It can then play new functions in traditional liposome (Liposome) and lipid nanoparticle (LNP) related fields, such as mRNA vaccines, nucleic acid delivery carriers, and drug delivery, thereby enabling the development of combination therapies in scientific research and industrial applications.
  • Liposome liposome
  • LNP lipid nanoparticle
  • lipid nanoparticles lipid hybrid substances
  • This invention greatly expands the current application scope of lipid nanoparticles and It provides fundamental knowledge for the fields of TPD and nanodelivery, and can in principle degrade a variety of human disease-related extracellular/membrane-associated/intracellular proteins in vivo.
  • the lipid-based protein degradation tool is composed of the lipid hybrid substance at the core and the POI recognition group. Nanoparticles composed of groups located on the periphery for protein degradation.
  • the whole structure can be a granular structure, including a core and a periphery.
  • the core part can be a lipid hybrid substance, and the periphery can be multiple POIs connected to the lipid hybrid substance.
  • the recognition group is a layer of numerous POI recognition groups connected to the outer surface of the lipid hybrid substance, thus forming an overall lipid-based protein degradation tool.
  • the lipid-based protein degradation tool may further include a lipid-based protein degradation tool provided with a connecting member between the POI recognition group and the lipid hybrid substance. protein degradation tools.
  • the molecular weight of the connecting member is 0-1000kDa;
  • the connecting member is one of a polymer connecting arm and a lipid connecting arm; wherein, the structure of a lipid-based protein degradation tool containing a lipid connecting arm is shown in Figure 46.
  • the polymer linker includes hydrophilic polymers, hydrophobic polymers and amphiphilic polymers.
  • the lipid linking arm is an amphipathic lipid linking arm.
  • the present disclosure provides another structure of a lipid-based protein degradation tool. Similar to the basic structure, there is also a connecting component between the lipid hybrid substance and the POI recognition group. A combination of "lipid hybrid substance-linking component-POI recognition group" connected in sequence is formed, in which the linking component is provided with a coupling group that can be connected to the POI recognition group, so that the combination of the two can be realized.
  • the POI recognition group and the connection member constitute a set of connection units;
  • the lipid-based The protein degradation tool is composed of the lipid hybrid material located at the core, a plurality of sets of connecting members located in the middle layer and connected to the lipid hybrid material, and a set of connecting members located at the periphery and connected to the lipid hybrid material.
  • the lipid-based protein degradation tool with a multi-layer structure is composed of multiple sets of connecting units of the POI recognition group connected to the connecting member.
  • the spatial structure of the lipid-based protein degradation tool is that the core is a lipid hybrid substance (core), and its surface layer is connected with multiple connecting components (middle layer), and each connecting component They are all connected with a lipid hybrid substance (periphery), forming a granular structure with three inner and outer layers.
  • the whole body can theoretically be spherical or other shapes.
  • the molecular weight of the connecting member is 0-1000 kDa.
  • the connecting member is one of a polymer connecting arm and a lipid connecting arm.
  • the polymer linker includes hydrophilic polymers, hydrophobic polymers and amphiphilic polymers.
  • the lipid linking arm is an amphipathic lipid linking arm.
  • the connecting components are divided into two categories, including: polymer connecting arms and lipid connecting arms. Either one can be selected according to the synthesis needs and water solubility needs of the substance. Moreover, the mass connecting arms are amphipathic. lipid linker arm.
  • the hydrophilic polymer may include, but is not limited to: polyethylene glycol (PEG), polyethylene oxide (PEO), poly(ethylene glycol) methacrylate (POEG), poly(2-methacryloyl) Oxyethyl phosphoryl (PMPC), polycarboxylic acid betaine (PCB), dextran, hyaluronic acid, chitosan, ⁇ -cyclodextrin, hyperbranched polyglycidyl ether (HPG), polyN- (2-Hydroxypropyl)methacrylamide (PHPMA), polyhydroxyethyl methacrylate (PHEMA), polyacrylamide (PAM), polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), polymaleic anhydride (HPMA), polyquaternary ammonium salts and pharmaceutically acceptable polymer salts thereof, polyethylenimine (PEI), poly N,N-dimethylaminoethyl methacrylate (PDMAEMA), polylysine
  • Hydrophobic polymers may include: polylactic acid-co-glycolic acid (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polycarbonate (PMC) and its derivatives, glycolide/lactide/ Various combinations of caprolactone/carbonate and copolymers of components, polyurethane (PU), polyether ether ketone (PEEK), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), polyethylene (PE) ), polyphenylalanine and other types of hydrophobic polyamino acids and derivatives of the above polymers and their pharmaceutically acceptable salts.
  • PLGA polylactic acid-co-glycolic acid
  • PLA polylactic acid
  • PCL polycaprolactone
  • PMC polycarbonate
  • PU polyurethane
  • PEEK polyether ether ketone
  • PMMA polymethyl methacrylate
  • PMMA polyvinyl alcohol
  • PE polyethylene
  • Amphiphilic polymers include: PEG-PLGA, PEG-PCL, PEG-PLA, PEG-PMC, and various amphiphilic block polymers and derivatives of the above-mentioned hydrophilic polymer and hydrophobic polymer combinations, as well as the above-mentioned Various amphiphilic polymers and derivatives composed of hydrophilic polymers and hydrophobic molecules (such as lipids).
  • the lipid connecting arm at least includes two ends, one end is a lipophilic end that can be connected to the lipid hybrid substance, and the other end is a hydrophilic end;
  • the lipophilic end is a lipid molecule.
  • the connecting member is a lipid connecting arm
  • its structure may include a structure of multiple ends, but there are at least two ends, the lipophilic end (connected to the lipid hybrid substance) and the hydrophilic end, and the hydrophilic end
  • the lipid end is a lipid molecule.
  • Lipid molecules include: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids lipids), saccharolipids, polyketides, cationic lipids and ionizable lipids.
  • the lipid molecule may be DSPE (distearoylphosphatidylethanolamine), distearoylphosphatidylcholine (DSPC), 1,2-dimyristate glyceryl (DMG,1, 2-Dimyristoyl-sn-glycerol), 1,2-Dipalmitoyl glyceryl ester (DPG, 1,2-DIPALMITOYL-SN-GLYCEROL), 1,2-dipalmitoyl glyceryl ester (DPyG, 1,2-Diphytanoyl- sn-glycerol), diacylglycerol (DAG); triglyceride (TAG), 1,2-dipalmitoylglycerol (DPG), 1,1'-[(1R)-1-(hydroxymethyl)- 1,2-Ethylenediyl]octadecanoate (DSG), diaraicosanylphosphatidylcholine (DAPC), 1-palmitoyl
  • the lipid molecules can be PEGylated lipids, where the relative molecular weight of PEG is 2000.
  • the PEGylated lipids can include: PEG-DSPE (polyethylene glycol-distearoylphosphatidylethanolamine), PEG-DMG (polyethylene glycol-glycerol dimyristate), conjugates of the aforementioned lipid molecules and PEG, and pharmaceutically acceptable salts thereof.
  • the PEG includes PEG and PEG with a methoxy or other group at the end of the PEG.
  • amphiphilic polymer is a polymer with a chain or branched molecular structure, which has at least one hydrophilic molecular end and one hydrophobic molecular end;
  • the structure of the amphiphilic polymer is a chain or branched molecular structure. Because of the characteristics of its chain or branched molecular structure, it can have multiple branches and multiple chains, but it has at least two ends, one One is the hydrophilic end of the molecule, and the other is the hydrophobic end.
  • amphiphilic polymer has a linear molecular structure, with one end being a hydrophilic molecular end and the other end being a hydrophobic molecular end.
  • amphiphilic polymer is further limited to a linear molecular structure.
  • nanoparticles are also included, wherein the nanoparticles are coated with the lipid hybrid substance at the core of the lipid-based protein degradation tool;
  • the nanoparticles include hydrophilic particles, hydrophobic particles and inorganic nanoparticles
  • the particle size of the nanoparticles is 5-1000 nm.
  • a special structure is provided, that is, the nanoparticles are at the core, and the lipid hybrid substance wraps the nanoparticles to form a composite structure.
  • lipid-based protein degradation tools can include the following morphological structures:
  • a special structure that is, nanoparticles are at the core, and lipid hybrid substances are coated on the nanoparticles to form a composite structure.
  • the nanoparticles are coated with the lipid hybrid substance at the core of the lipid-based protein degradation tool
  • nanoparticles (nanoparticles coated in the core of the lipid hybrid substance) include:
  • Hydrophilic particles can be dendritic polymers, hyperbranched polymers, various nanogels composed of the aforementioned hydrophilic polymers and their derivatives, and nanoalbumin;
  • Hydrophobic particles can be polylactic acid-co-glycolic acid (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polycarbonate (PMC) and its derivatives, glycolide/ Copolymers of various combinations and components of lactide/caprolactone/carbonate, polyurethane (PU), polyether ether ketone (PEEK), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), Nanoparticles prepared from polyethylene (PE), polyphenylalanine and other types of hydrophobic polyamino acids and their derivatives;
  • PLGA polylactic acid-co-glycolic acid
  • PLA polylactic acid
  • PCL polycaprolactone
  • PMC polycarbonate
  • PU polyurethane
  • PEEK polyether ether ketone
  • PMMA polymethyl methacrylate
  • PVA polyvinyl alcohol
  • 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, and aluminum oxide nanoparticles , titanium oxide nanoparticles, aluminum-iron alloy particles, and titanium-iron alloy particles, all of which are materials with stable and low biological toxicity; (4) and mixed nanoparticles composed of the aforementioned nanoparticles.
  • the nanoparticles when they are either hydrophilic particles or hydrophobic particles and are polymers, they can also be called lipid polymers (LPP, lipopolyplex) after being coated with lipid hybrid substances. That is, the surface of nanoparticles with hydrophilic or hydrophobic polymers is coated with a lipid film, and cationic nanoemulsion (Cationic nanoemulsion), that is, emulsion droplets composed of cationic lipids, has also been widely studied and applied in vaccines and drug delivery.
  • LPP lipopolyplex
  • the antibody is a therapeutic monoclonal antibody, a multispecific antibody, a nanobody, and the aforementioned antibody derivatives or antibody-conjugated drugs;
  • the polypeptide is a polypeptide with POI specific binding ability
  • the small molecule is a compound with POI specific binding ability
  • the small molecules include derivatives of the CDK4/6 protein inhibitor Palbociclib, derivatives of the BRD4 protein inhibitor JQ1, derivatives of the ⁇ -Amyloid protein probe AV-45, and Pittsburgh compounds.
  • polypeptides include binding polypeptides of ACE2, CD13, and ⁇ -Amyloid;
  • the antibodies include: CTX, NTZ, PTZ, CRLZ, INE, ATZ, and aducanumab, Miltuximab.
  • the present disclosure also provides a lipid-based protein degradation tool for use in the preparation of drugs, vaccines and delivery systems for the treatment and prevention of diseases related to abnormal protein accumulation; wherein the abnormal protein accumulation diseases include tumors, immune Systemic diseases, inflammation and pathogenic infections, neurodegenerative diseases, hematological diseases and metabolic diseases.
  • abnormal protein accumulation diseases include tumors, immune Systemic diseases, inflammation and pathogenic infections, neurodegenerative diseases, hematological diseases and metabolic diseases.
  • the present disclosure also provides a lipid-based protein degradation tool for use in the preparation of detection products and/or kits for abnormal protein accumulation related diseases and protein interaction research; wherein, the abnormal protein accumulation disease Including tumors, immune system diseases, inflammation and pathogenic infections, neurodegenerative diseases, hematological diseases and metabolic diseases.
  • the abnormal protein accumulation disease Including tumors, immune system diseases, inflammation and pathogenic infections, neurodegenerative diseases, hematological diseases and metabolic diseases.
  • the present disclosure also provides a method for preparing a lipid-based protein degradation tool, wherein the lipid-based protein degradation tool is a combination of the POI recognition group and the lipid hybrid substance.
  • protein degradation tools there are two preparation methods:
  • the POI recognition group is covalently bonded to the lipid hybrid substance based on the coupling group, thereby forming the lipid-based protein degradation tool.
  • the lipid-based protein degradation tool also includes a protein degradation tool composed of the POI recognition group connected to the lipid hybrid substance through a connecting member;
  • the preparation method is:
  • the POI recognition group is modified on the surface of the nanoparticle, or the POI recognition group is connected to the connecting arm, or the POI recognition group is modified on the connecting arm and connected to the surface of the nanoparticle through the connecting arm.
  • NP nanoparticles
  • the POI recognition group is assembled, Exposed to the outside of nanoparticles or connecting arms, where nanoparticles (NP, Nanoparticles) can penetrate cells independently of specially designed receptor-ligand matching pairs, and can be coupled to small molecules, peptides, nucleic acid aptamers, POI recognition groups such as antibodies specifically carry out protein hijacking and targeted degradation, thereby realizing the assembly of nanoparticle-based TPD tools (TPD-NP).
  • TPD-NP nanoparticle-based TPD tools
  • This convenient nanoparticle-based TPD tool has easy access to drug loading and tissue-specific targeting capabilities, making possible combined drug and protein degradation therapy as well as translational/precision medicine.
  • the invention of TPD-NP and the exploration of its mechanism have greatly expanded the scope of TPD tools and provided fundamental knowledge for the fields of TPD and nanodelivery, And in principle, it can degrade a variety of human disease-related extracellular/membrane-associated/intracellular proteins in vivo.
  • the disclosed nano-protein degradation tool has a flexible structure, is easy to transform, and can obtain the ability to carry drugs, target, and cross biological barriers.
  • the disclosed nano-protein degradation tool has universal applicability, the target can be replaced at will, all three components can be replaced, and the application scenarios are wide. All components of the disclosed nano-protein degradation tool can be clinically approved materials, with high in vivo application potential and translational value.
  • the disclosed nano-protein degradation tool does not need to be synthesized from scratch and is a ready-to-use platform, which greatly reduces the complexity of development and production. sex and difficulty.
  • TPD-NP can degrade extracellular/intracellular/membrane proteins.
  • extracellular/membrane protein degradation tools such as LYTAC
  • TPD-NP does not require additional design of structures to assist the hijacked protein in being endocytosed.
  • the nano-protein degradation tool of the present disclosure does not require a special structure to guide protein degradation.
  • Nanoparticles can be loaded with drugs, can be designed for controlled release, can be designed into synergistic treatment materials such as photothermal magnetism, etc., and can be used for imaging, contrast, and protein degradation for synergistic treatment and integration of diagnosis and treatment.
  • the body fluid stability of NP can reduce drug loss and improve drug potential.
  • the present disclosure achieves target protein degradation based on lipid hybrid substances by modifying the POI recognition group on the surface of the lipid hybrid substance, and exposing the POI recognition group to the outside of the lipid hybrid substance after assembly. It greatly reduces the difficulty of synthesizing protein degradation tools.
  • a large number of compounds, peptides, antibodies and nucleic acid aptamers that have binding ability to target proteins can be upgraded into protein degradation drugs, and then It can play new functions in traditional liposome and lipid nanoparticle (LNP) related fields, such as mRNA vaccines, nucleic acid delivery carriers, and drug delivery, thereby enabling the development of combination therapies in scientific research and industrial applications.
  • LNP liposome and lipid nanoparticle
  • lipid nanoparticles lipid hybrid substances
  • This invention greatly expands the current application scope of lipid nanoparticles and It provides fundamental knowledge for the fields of TPD and nanodelivery, and can in principle degrade a variety of human disease-related extracellular/membrane-associated/intracellular proteins in vivo.
  • the first degradation tool was prepared, including coupling a POI recognition group to a connecting arm and then self-assembly.
  • the POI recognition group is the monoclonal antibody drug Nitrozine (NTZ)
  • the connecting arm is NHS-PEG-DSPE (N-hydroxysuccinimide-modified polyethylene glycol-distearoylphosphatidylethanolamine, or DSPE-PEG-NHS).
  • Pretreatment Replace the original antibody buffer with phosphate buffered saline PBS.
  • the EGFR monoclonal antibody Nimotuzumab (NTZ) which has been used in clinical treatment, is used.
  • the nimotuzumab solution centrifuge it at 4000 ⁇ g for 2 minutes with a 3kDa ultrafiltration tube, and then concentrate the antibody, and then dilute it with PBS; after repeated concentration and dilution three times, the main component of the buffer is replaced with PBS, and the antibody solution is diluted , the protein concentration was determined by absorbance at A280 in IgG mode of the NanoDrop one C instrument, and its results were verified by the BCA protein quantification kit.
  • coupling methods include but are not limited to non-directed coupling and fixed-site coupling.
  • Non-site-directed coupling includes amino coupling, carboxyl coupling, and bridged thiol coupling.
  • Site-directed coupling includes click chemical reaction, selenium bond coupling, serine coupling, cysteine coupling, unnatural amino acid coupling, enzyme catalyzed post-coupling, and sugar site coupling.
  • Figure 4 of the present disclosure illustrates the synthesis process of this embodiment.
  • the antibody and NHS-PEG-DSPE undergo a coupling reaction through the amino group on the antibody and the NHS group at the end of DSPE-PEG to obtain the conjugate NTZ-PEG-DSPE.
  • Figure 5 of the present disclosure shows the Coomassie Brilliant Blue staining results of this example.
  • the Coomassie Brilliant Blue staining results show that after the NTZ antibody reacts with the connecting arm to obtain the nanoparticle NTZ-PEG-DSPE, the mass increases and the migration rate slows down. After Coomassie Brilliant Blue binds to the protein, the color band is higher, indicating that the POI recognition base The group was successfully attached to the PEG-DSPE arm.
  • Figure 6 of the present disclosure shows a Western blot protein electrophoresis pattern of the protein degradation effect of this embodiment. The results show that NTZ-PEG-DSPE nanoparticles can effectively degrade the target protein EGFR.
  • the volume of cell culture medium in all examples is the same, and the molar concentration units of the treatments received are ⁇ M ( ⁇ mol/L) and nM (nmol/L).
  • the molar concentrations used in all control groups are consistent with the degradation tool group.
  • the molar concentration and volume of the antibody NTZ in the NTZ group and the NTZ-NP group are the same.
  • the lipid impurities in the NP group without antibodies and the NTZ-NP group are the same.
  • the molar concentration and volume of chemical substances are the same.
  • the NP group refers to the nanoparticles that are not connected to POI recognition groups in the embodiment.
  • control POI recognition group group or the control lipid hybrid substance group is treated at an equal molar concentration of the highest concentration treatment in the concentration test group.
  • All protein electropherograms use GAPDH or VIN (vinculin) as internal reference.
  • a second degradation tool was prepared, including connecting a POI recognition group to a connecting arm, and then self-assembling to form the second degradation tool.
  • the POI recognition group is exposed outside the bulk.
  • the POI recognition group is the amino derivative JQ1-NH 2 of the inhibitor JQ1 of the BRD4 protein, whose amino group is used for nanoparticle coupling, and BRD4 is a tumor epigenetic and proliferation regulatory molecule.
  • the POI recognition group is EGFR monoclonal antibody Nitrozumab NTZ, and its amino group is used for coupling of nanoparticles.
  • the nanoparticles are nanoparticles formed by NHS-PEG-PLGA (N-hydroxysuccinimide-polyethylene glycol-polylactic acid-glycolic acid copolymer) organic phase transferred to aqueous phase, NHS exposed to nanoparticle exterior.
  • NHS-PEG-PLGA N-hydroxysuccinimide-polyethylene glycol-polylactic acid-glycolic acid copolymer
  • coupling methods include but are not limited to non-directed coupling and fixed-site coupling.
  • Non-directed coupling such as amino coupling, carboxyl coupling, and bridged thiol coupling.
  • Site-directed coupling includes click chemical reaction, selenium bond coupling, serine coupling, cysteine coupling, unnatural amino acid coupling, enzyme catalyzed post-coupling, and sugar site coupling.
  • the POI recognition group selected the amino derivative JQ1-NH 2 of the BRD4 protein inhibitor JQ1, and was connected to the PEG terminal NHS group of the PEG-PLGA nanoparticles through JQ1-NH 2 .
  • the PEG-PLGA nanoparticles used are self-assembled into nanoparticles by transferring the organic phase of the polymer NHS-PEG(3kDa)-PLGA(5kDa) to the aqueous phase.
  • the concentration was measured using the absorption standard curve correction of the UV absorption peak of the compound.
  • M231 human breast cancer tumor cells were treated with JQ1 at a final concentration of 500 nM, 1 ⁇ M, and 1.5 ⁇ M, a molar amount of JQ1-NP equivalent to JQ1, and a molar amount of NP equivalent to JQ1 for 24 hours, and then the total cell protein was collected and the protein was detected.
  • JQ1 at a final concentration of 500 nM, 1 ⁇ M, and 1.5 ⁇ M
  • NTZ monoclonal antibody was selected as the POI recognition group, and the amino group of the lysine residue of the NTZ monoclonal antibody was coupled to the PEG terminal NHS group of the PEG-PLGA nanoparticles to obtain NTZ-NP2.
  • the PEG-PLGA nanoparticles used are self-assembled into nanoparticles by transferring the organic phase of the polymer NHS-PEG(3kDa)-PLGA(5kDa) to the aqueous phase.
  • the concentration was determined using the IgG mode of the Nanodrop one C instrument with absorbance measurement at A280 and the BCA protein quantification kit to verify the results.
  • Figure 7 of the present disclosure is a schematic diagram of the synthesis process of the second degradation tool.
  • the structural formula in Figure 8 of the present disclosure is the structure of JQ1- NH2 , whose terminal free NH2 is used for coupling with PEG-PLGA nanoparticles.
  • the dynamic light scattering DLS results in Figure 8 show that the nanoparticle size is approximately 100 nm.
  • the average dispersion coefficient PDI is about 0.2, indicating that the nanoparticles are relatively uniform.
  • Figure 9 of the present disclosure shows the electrophoresis results of the second degradation tool JQ1-NP protein in Example 2. Protein electrophoresis results showed that after JQ1 was coupled to nanoparticles, the cellular BRD4 protein level was down-regulated, while its solvent PBS control group, pure JQ1 small molecule group, and pure nanoparticle control group had no BRD4 degradation effect.
  • Figure 10 of the present disclosure shows the electrophoresis results of the second degradation tool NTZ-NP2 protein in Example 2. Protein electrophoresis results showed that after treatment with 500nM NTZ antibody-conjugated nanoparticles NTZ-NP2 for 24 hours, the EGFR protein level of M231 cells was down-regulated, while its solvent PBS control group, pure EGFR monoclonal antibody group, and pure nanoparticle control group had no EGFR degradation effect.
  • the third degradation tool was prepared, including coupling the POI recognition group to the connecting arm and then coupling it to the nanoparticles to form a composite structure.
  • the POI recognition group is NTZ antibody.
  • the NTZ-PEG-DSPE coupling intermediate is obtained by coupling the amino group on the antibody to the NHS-PEG-DSPE linking arm, and then the NTZ-PEG-DSPE coupling intermediate is precipitated on the surface of the PLGA nanoparticles to produce a composite structure, which finally forms A protein degradation tool with an inner layer of nanoparticle core (PLGA), a middle layer of amphiphilic block polymer (PEG-DSPE), and an outer layer of NTZ antibody, namely "NTZ-NP", was developed.
  • PLGA nanoparticle core
  • PEG-DSPE middle layer of amphiphilic block polymer
  • NTZ-NP an outer layer of NTZ antibody
  • the coupling method of the POI recognition group and the connecting arm PEG-DSPE adopts the coupling method in the first degradation tool.
  • the POI recognition group-PEG-DSPE is coupled with PLGA nanoparticles, and the PLGA organic phase is transferred to the aqueous phase.
  • the POI recognition group-PEG-DSPE self-assembles with PLGA in the aqueous phase and precipitates on the core surface of PLGA.
  • Figure 11 of the present disclosure illustrates the synthesis process of the third degradation tool.
  • the coupling intermediate is obtained by coupling the amino group on the antibody to the connecting arm, and then the coupling intermediate is precipitated on the surface of the nanoparticle to generate a composite structure.
  • Figure 12 of the present disclosure shows the morphological structure of the third degradation tool prepared in this embodiment, which is that the inner layer is a nanoparticle core (PLGA), the middle layer is an amphiphilic block polymer (PEG-DSPE), The periphery is a protein degradation tool for NTZ antibodies.
  • PLGA nanoparticle core
  • PEG-DSPE amphiphilic block polymer
  • the periphery is a protein degradation tool for NTZ antibodies.
  • Figure 13 of the present disclosure shows the dynamic light scattering results and transmission electron micrograph of the third degradation tool prepared in this embodiment.
  • the diameter of the nanoparticles was measured by dynamic light scattering to be approximately 150 nm, and the average dispersion coefficient (PDI) was 0.153, indicating that the obtained nanomedicine has good uniformity.
  • the morphology of the nanoparticles measured by transmission electron microscopy is spherical as shown in the figure, and the scale bar is 100 nm. The above results prove that the third degradation tool was successfully obtained.
  • NTZ is a human EGFR monoclonal antibody
  • EGFR is a tumor proliferation signaling receptor and surface marker.
  • NTZ-NP was treated with MDA-MB-231 (M231) human breast cancer cells, HeLa human cervical cancer cells, and U87 human glioma cells at a final concentration of 500 nM for 24 hours, and then EGFR protein expression was detected.
  • Figure 14 of the present disclosure shows the protein electrophoresis results of the third degradation tool (NTZ-NP) in Example 3 on the protein degradation effect of human breast cancer M231 cells, human cervical cancer HeLa cells, and human brain glioma U87 cells.
  • NTZ-NP third degradation tool
  • Figure 15 of the present disclosure shows the immunofluorescence staining results of M231 cells after being treated with the third degradation tool.
  • M231 cells were treated with 500nM antibody dose of NTZ-NP for 24 hours, the expression of EGFR on the cell surface was detected by immunofluorescence staining and confocal microscopy.
  • Figure 16 of the present disclosure shows the degradation results of green fluorescently labeled EGFR protein taken in a fixed field of view live cell time series after M231 cells were treated with the third degradation tool.
  • M231 cells transfected with EGFP green fluorescent protein fused to the intracellular domain of EGFR (before the stop codon) for experiments.
  • Figure 17 of the present disclosure shows the cell viability results of CCK8 detection of HepG2 cells after being treated with the third degradation tool. As shown in Figure 17, after human liver cancer HepG2 cells were treated with 500nM antibody dose of NTZ-NP for 24 hours, the cell proliferation was significantly inhibited as detected by the CCK8 cell viability assay kit. ***p ⁇ 0.001 has statistically significant difference.
  • Figure 18 of the present disclosure shows the EGFR protein expression of the third degradation tool in Example 3, in which picture a shows the EGFR protein expression after 24 hours of treatment with different concentrations of NTZ-NP. The results show that EGFR The concentration that can be effectively degraded can be as low as 10nM, which theoretically has good druggability.
  • Picture b shows the total protein collected from M231 cells treated with 500nM NTZ-NP for different times for protein electrophoresis. Starting from 24 hours, the medium was replaced with fresh medium without treatment in order to observe the dynamic recovery of the target protein. It can be seen that the optimal degradation time for a single treatment is 24-48 hours, and it gradually recovers after 72 hours. The performance of gradual recovery is consistent with the theoretical basis of "temporary degradation" of protein degradation tools.
  • Experiment 4 method Adjust the proportion of NTZ-PEG-DSPE by mixing different proportions of PEG-DSPE linking arms without antibodies during the self-assembly of NTZ-PEG-DSPE and PLGA, and then 500nM (equivalent to NTZ M231 cells were treated with different NPs (molar concentrations of antibodies) for 24 h.
  • Figure 19 of the present disclosure shows the expression of EGFR protein of the third degradation tool in Example 3, which studies the coupling intermediate composed of a POI recognition group and a connecting arm and the coupling intermediate without POI recognition.
  • the results show that under the same antibody content, when the ratio of NTZ-PEG-DSPE intermediate to blank connecting arm is 1:1, that is, when the content of NTZ-PEG-DSPE coupling intermediate is about 50%, the protein degradation effect is better.
  • the above results show that compared with the experimental group of 50% NTZ-PEG-DSPE arm, the experimental group of 100% NTZ-PEG-DSPE arm may cause steric hindrance due to the POI recognition group NTZ, which in turn affects protein degradation.
  • the third degradation tool was prepared, including coupling the POI recognition group to the connecting arm and then coupling it to the nanoparticles to form a composite structure.
  • the POI recognition group is: NTZ antibody
  • the connecting arm is: polyethylene glycol PEG
  • the nanoparticles are a composite structure
  • the nanoparticles are inorganic material gold nanoparticles (AuNP), thus forming a composite structure, namely "NTZ-AuNP" ".
  • the coupling method of the POI recognition group and the linking arm NHS-PEG-SH adopts the coupling method in the first degradation tool.
  • the POI recognition group-PEG-DSPE is coupled with gold nanoparticles through the spontaneous formation of gold-sulfur bonds between sulfhydryl groups and gold nanoparticles.
  • Coupling methods include covalent reactions via reactive groups or via non-covalent coupling.
  • Prepare 1mg/mL NHS-PEG ( 2kDa )-SH solution in 0.1M, pH 8.0 NaHCO3 solution, and immediately add nimotuzumab at a molar ratio of 1:1, stir at 600rpm on ice, dropwise PEG was added and incubated overnight at 4°C with stirring to obtain the NTZ-PEG-SH connecting arm.
  • AuNP and NTZ-PEG-SH are coupled through a gold-sulfur bond reaction between -SH and gold. Specifically, stir in PBS at room temperature 600 rpm for 2 hours, and then react overnight at 4°C. It is then concentrated by ultrafiltration. Protein concentration was measured by NanoDrop one C (Thermofisher) in IgG mode with absorbance at A280 and verified by BCA protein assay kit.
  • Figure 20 of the present disclosure shows a schematic structural diagram of the third degradation tool (NTZ-AuNP) in Example 4.
  • Figure 21 of the present disclosure shows the electrophoresis results of the protein degradation effect of the third degradation tool (NTZ-AuNP) in Example 4. As shown in Figure 21, after M231 cells were treated with NTZ-AuNP and control for 24 hours, the expression of EGFR protein was significantly reduced.
  • the third degradation tool was prepared, including coupling the POI recognition group to the connecting arm and then coupling it to the nanoparticles to form a composite structure.
  • the POI recognition group is: a polypeptide with CD13 protein or ACE2 protein or ⁇ -amyloid 1-42 oligomer binding ability
  • the connecting arm is: amphiphilic polymer (PEG-DSPE)
  • the nanoparticle is PLGA, so Constitute a composite structure, namely "CD13-NP” or "ACE2-NP” or "AB-NP”.
  • CD13 is a receptor for the coronavirus that causes the common cold and a tumor marker.
  • Its binding polypeptide AP-1 is from the literature, and its sequence is NH 2 -YVEYHLC-COOH.
  • ACE2 is a coronavirus receptor.
  • the COVID19 virus SARS-Cov2 infects cells through the ACE2 protein receptor.
  • Its binding polypeptide sequence NH 2 -CSPLRYYPWWACT-COOH comes from the literature.
  • ⁇ -amyloid 1-42 oligomer ( ⁇ -amyloid oligomer) is a potential pathogenic protein of Alzheimer's disease. It is an extracellular protein. Its precursor is expressed on the cell membrane.
  • oligomers After cleaving, it is secreted extracellularly to form oligomers. bodies and fibers, among which oligomers are considered to be the most toxic. As the accumulation of extracellular protein ⁇ -amyloid oligomer brings pathological risks such as neuroinflammation, its binding polypeptide sequence NH 2 -KLVFF-COOH is derived from the literature.
  • the coupling method of polypeptides is the coupling method of general antibody conjugated drugs, including but not limited to site non-specific coupling and specific coupling, as well as the most common amino coupling, click chemical reaction, carboxyl coupling, thiol coupling Coupling, selenium bond coupling.
  • Figure 22 of the present disclosure shows the hydrogen nuclear magnetic resonance spectrum ( 1 H NMR) of the POI recognition group and connecting arm connection product (ACE2-PEG-DSPE) in the third degradation tool (ACE2-NP) in this example.
  • Figure 23 of the present disclosure shows the hydrogen nuclear magnetic resonance spectrum ( 1H NMR) of the POI recognition group and connecting arm connection product (CD13-PEG-DSPE) in the third degradation tool (CD13-NP) in this example.
  • Figure 24 of the present disclosure shows the results of flow cytometry detection of GFP after the third degradation tool (ACE2-NP) in this embodiment treated 293 cells labeled with ACE2 by GFP.
  • ACE2-NP third degradation tool
  • Figure 25 of the present disclosure shows the protein electrophoresis results after the third degradation tool (CD13-NP) treated human liver cancer cell HepG2 in this embodiment. As shown in Figure 25, after treatment with 2 ⁇ M CD13-binding peptide (CD13) or its NP conjugate (CD13-NP) for 24 hours, the expression of CD13 in HepG2 cells was significantly reduced.
  • CD13-NP third degradation tool
  • Figure 26 of the present disclosure shows the intracellular endocytosis of FITC green dye-labeled ⁇ -amyloid 1-42 oligomers detected by confocal microscopy after human glial cells were treated with the third degradation tool (AB-NP) in this example.
  • experimental results 1 ⁇ M FITC fluorescein-labeled 5 ⁇ M ⁇ -amyloid oligomer oligomers and AB-NP were co-cultured in human glial cells for 4 hours, and then the extracellular solution was washed away with PBS.
  • the preparation of the third degradation tool includes coupling the POI recognition group to the connecting arm and then coupling it to the nanoparticles to form a composite structure.
  • the POI recognition group is the small molecule drug Palbociclib or the small molecule probe AV-45
  • the connecting arm is an amphiphilic polymer (PEG-DSPE)
  • the nanoparticle is PLGA.
  • the POI recognition group is the CDK4 inhibitor Palbociclib, and its nanoparticle conjugate is Palb-NP.
  • CDK4 is a cyclin-dependent kinase that is widely involved in cell senescence and tumorigenesis.
  • Palb-PEG-DSPE-(PLGA), that is, Palb-NP is synthesized by PLGA organic phase-to-aqueous phase self-assembly.
  • the method is the same as in Example 3.
  • PLGA is dissolved in DMF
  • Palb-PEG-DSPE is dissolved in in PBS.
  • the POI recognition group is ⁇ -amyloid probe AV-45, and its nanoparticle conjugate is AV45-NP.
  • ⁇ -amyloid is a potential pathogenic protein of Alzheimer's disease. It is an extracellular protein. Its precursor is expressed on the cell membrane. After being cleaved, it is secreted extracellularly to form oligomers ( ⁇ -amyloid 1-42oligomer) and fibers. Among them, oligomers are considered to be the most toxic. As the accumulation of extracellular protein ⁇ -amyloid oligomers brings pathological risks such as neuroinflammation, isotope-labeled AV-45 is a clinically commercial ⁇ -amyloid probe.
  • AV-45-SH has blue autofluorescence, which facilitates cell tracking, and its sulfhydryl SH group is used for coupling with Mal-PEG-DSPE.
  • Preparation method Combine AV45-SH with thiol group and Mal-PEG2K-DSPE, and then combine with PLGA.
  • AV45-NP Dialyze in the dark for 24 hours to remove free AV45-SH, and then connect the PLGA core.
  • the connection method adopts the method of Experiment 1 of this example.
  • the PLGA organic phase is transferred to the aqueous phase for self-assembly.
  • AV45-PEG- DSPE self-assembles with PLGA in the aqueous phase. Quantitation was performed by UV absorption of AV45.
  • ⁇ -amyloid hijacking experiment oligomers prepared from ⁇ -amyloid 1-42 polypeptide labeled with 10 ⁇ M FITC green dye were co-incubated with 10 ⁇ M AV45-NP, and then the processed substances that did not enter the cells were washed away with PBS. Perform confocal microscopy imaging to detect the green fluorescence of ⁇ -amyloid 1-42 oligomers. Strong green fluorescence indicates that more hijacked cells have been hijacked. Theoretically, more phagocytosis and lysosomal transfer can lead to better results. To reduce the toxicity caused by the accumulation of extracellular ⁇ -amyloid oligomer, the lysosomal dye lysoTracker is used to mark lysosomes.
  • the concentration was measured using NMR to calculate the grafting rate and corrected using the UV absorption standard curve.
  • Figure 27 of the present disclosure shows the hydrogen nuclear magnetic resonance spectrum ( 1 H NMR) of the POI recognition group and connecting arm connection product (Palb-PEG-DSPE) in the third degradation tool (Palb-NP) in Example 6.
  • the proton nuclear magnetic resonance spectrum in Figure 27 shows that the small molecule drug Palbociclib was successfully coupled to PEG-DSPE.
  • Figure 28 of the present disclosure shows a protein electrophoresis pattern of the protein degradation effect of the third degradation tool (Palb-NP) in Example 6.
  • M231 cells were treated with 2.5 ⁇ M Palbociclib or Palbociclib-conjugated nanoparticles (Palb-NP) for 24 hours, and the protein expression was detected to detect CDK4 protein expression.
  • the results in Figure 28 show that Palb-NP can effectively inhibit tumor proliferation.
  • Figure 29 of the present disclosure shows the experimental results of crystal violet staining colony formation after M231 cells were treated with the third degradation tool in Example 6. M231 cells were treated with 3.5 ⁇ M Palbociclib or Palb-NP for 48 hours, and then cultured for 7 days to perform crystal violet staining and colony formation experiments. The results in Figure 29 show that Palb-NP inhibits tumor proliferation better than Palbociclib.
  • Figure 30 of the present disclosure is a schematic diagram of the structure of AV45-NP.
  • Figure 31 of the present disclosure shows the hydrogen nuclear magnetic resonance spectrum ( 1 H NMR) of the POI recognition group and connecting arm connection product (AV45-PEG-DSPE) in the third degradation tool (AV45-NP) in Example 6, Figure
  • the proton nuclear magnetic resonance spectrum in 31 showed that the small molecule drug ⁇ -amyloid probe AV-45 was successfully coupled to PEG-DSPE.
  • Figure 32 of the present disclosure shows the intracellular endocytosis of FITC green dye-labeled ⁇ -amyloid 1-42 oligomers detected by confocal microscopy after human glial cells were treated with the third degradation tool (AV45-NP) in Example 6. experimental results. Human glial cells were treated by co-incubating 10 ⁇ M AV-45 or AV45-NP with 10 ⁇ M FITC green fluorescent dye-labeled ⁇ -amyloid oligomers in culture medium for 12 hours.
  • the third degradation tool was prepared to obtain CTX-NP, PTZ-NP, ATZ-NP, CRLZ-NP and NTZ/INE-NP respectively, including coupling the POI recognition group to the connecting arm, Then coupled with nanoparticles to form a composite structure.
  • the POI recognition group, connecting arm and nanoparticles are PLGA, as shown in Table 6 respectively.
  • Preparation method Synthesize CTX-PEG-DSPE, PTZ-PEG-DSPE, ATZ-PEG-DSPE, CRLZ-PEG-DSPE and INE-PEG-DSPE according to the preparation method provided in Example 1, and use the method in Example 3
  • the preparation method synthesizes CTX-NP, PTZ-NP, ATZ-NP, CRLZ-NP and INE-NP.
  • NTZ/INE-NP is self-assembled in PLGA by equimolar amounts of NTZ-PEG-DSPE and INE-PEG-DSPE. Obtained, its structure is shown in Figure 8.
  • Figure 33 of the present disclosure shows a protein electrophoresis pattern of the protein degradation effect of the third degradation tool (CTX-NP) in Example 7. As shown in Figure 33, after 500nM CTX-NP treated M231 cells for 24 hours, the protein electrophoresis results showed that the expression of the receptor protein EGFR was significantly reduced.
  • CTX-NP third degradation tool
  • Figure 35 of the present disclosure shows the protein electrophoresis pattern of the protein degradation effect of the third degradation tool (ATZ-NP) in Example 7. As shown in Figure 35, after 500nM ATZ-NP treated 293T cells overexpressing PD-L1 for 24 hours, the protein electrophoresis results showed that the expression of PD-L1 receptor protein was significantly reduced.
  • Figure 36 of the present disclosure shows the protein electrophoresis pattern of the protein degradation effect of the third degradation tool (CRLZ-NP) in Example 7. As shown in Figure 36, after 500nM CRLZ-NP treated JURKAT cells overexpressing PD-1 for 24 hours, the protein electrophoresis results showed that the expression of PD-1 receptor protein was significantly reduced.
  • CRLZ-NP third degradation tool
  • Figure 37 of the present disclosure shows the protein electrophoresis pattern of the protein degradation effect of the third degradation tool (INE/NTZ-NP) in Example 7. As shown in Figure 37, after 293T cells expressing HER2 were treated with 500nM INE-NP or NTZ/INE-NP for 24 hours, the protein electrophoresis results showed that the expression of HER2 and EGFR receptor proteins was significantly reduced. This shows that the present disclosure has the ability to obtain multi-specific targeting through a simple "plug and play" approach and has good application potential.
  • INE/NTZ-NP third degradation tool
  • Experiment 1 In vivo therapeutic effect of NTZ-NP on the MDA-MB-231 breast cancer subcutaneous tumor transplant model in animals.
  • This example uses the optional third degradation tool obtained in Example 3, in which the POI recognition group is NTZ, the connecting arm is DSPE-PEG, and the nanoparticle core is PLGA.
  • NTZ-NP the in vivo therapeutic effect of NTZ-NP on the MDA-MB-231 (M231) breast cancer subcutaneous tumor transplant model was explored.
  • administration 15 mg/kg was started every other day, and the volume of the mouse tumors was continuously monitored (tumor length a width b, volume calculation formula: 1/2ab 2 ) , and weight changes.
  • a total of 7 times the mice were euthanized the day after the last administration, and the tumor tissues were isolated, and the tumor tissues were subjected to protein electrophoresis and immunofluorescence detection.
  • Figure 38a of the present disclosure shows the volume of nude mouse tumors during NTZ-NP treatment of the M231 cell nude mouse subcutaneous tumor model.
  • the experimental results show that compared with the solvent control PBS group, the equimolar amount of NTZ antibody group and the equimolar amount of nanoparticles without antibody group, the tumor volume proliferation was significantly inhibited in the NTZ-NP group.
  • Figure 38b shows the EGFR protein expression level detected by protein electrophoresis after separation of tumor tissue. It can be seen that EGFR is significantly down-regulated.
  • the complete tumor tissues of each treatment were taken from 5 different experimental mice and subjected to protein electrophoresis and grayscale statistics relative to their own internal controls. The statistical results *p ⁇ 0.05 showed statistical differences.
  • Figure 38c shows the changes in mouse body weight during NTZ-NP treatment. The results show that there is no significant change in body weight.
  • Figure 39 shows the results of immunofluorescence staining to detect EGFR after tumor tissue was isolated after NTZ-NP treatment.
  • Figure 40 shows the results of separating tumor tissue after NTZ-NP treatment and performing immunofluorescence staining to detect apoptosis markers.
  • the tumor tissue was separated and paraffin-embedded into sections, followed by immunofluorescence staining to detect EGFR and cleaved caspase-3, the core apoptosis signal.
  • the results of immunofluorescence staining showed that the EGFR signal decreased and the apoptosis signal increased in the NTZ-NP group.
  • the results show that the NTZ-NP group of nanoparticles can degrade the growth factor receptor protein EGFR on the surface of mouse tumor cells.
  • NTZ-NP has a therapeutic effect on mouse subcutaneous tumors, showing potential for in vivo application.
  • This example uses the optional third degradation tool obtained in Example 3, in which the POI recognition group is NTZ, the connecting arm is DSPE-PEG, and the nanoparticle core is PLGA.
  • the POI recognition group is NTZ
  • the connecting arm is DSPE-PEG
  • the nanoparticle core is PLGA.
  • the acute liver and kidney toxicity of NTZ-NP nanoprotein degradation tools in mice was explored.
  • the nanoparticles and the solvent were compared with PBS, an equimolar amount of NTZ antibody, and an equimolar amount of NP without NTZ antibody (10 mg/kg), and were injected into white mice through the tail vein. Blood was taken 12 hours after the injection, and the blood was collected through the kit. Conduct blood biochemical index testing.
  • Figure 41 of the present disclosure shows the detection of blood, liver and kidney function indicators in white mice after a single administration of the third degradation tool (NTZ-NP) in Example 8.
  • the experimental results show that plasma urea (BUN), uric acid (UA), creatinine (CR), albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT) and Tianmen
  • plasma urea BUN
  • uric acid U
  • CR creatinine
  • ALP alkaline phosphatase
  • ALT alanine aminotransferase
  • Tianmen There were no obvious abnormalities in related indicators such as aspartate aminotransferase (AST), indicating that the nanoparticles had no obvious toxic side effects on liver and kidney metabolism in mice.
  • This example uses the optional third degradation tool obtained in Example 3, in which the POI recognition group is NTZ, the connecting arm is DSPE-PEG, and the nanoparticle core is PLGA.
  • the near-infrared dye DIR which cannot pass through the blood-brain barrier and cannot target tumor cells, is wrapped in a PLGA core (after DIR and PLGA are organically dissolved, they are added dropwise into the NTZ-PEG-DSPE aqueous solution to self-assemble, and then purified Concentrate) to obtain DIR-loaded NTZ-NPs.
  • Ang-NTZ-NP the angiopep-2 polypeptide is a representative blood-brain barrier crossing polypeptide and tumor-targeting polypeptide, and its polypeptide sequence (NH 2 -TFFYGGSRGKRNNFKTEEY-COOH) is derived from the literature.
  • Inject equal molar amounts of two nanoparticles (antibody amount 10 mg/kg, DIR amount 0.25 mg/kg) intravenously into tumor-bearing (human brain glioma orthotopic transplantation) nude mice, and compare DIR through the small animal imager IVIS system Fluorescence is used to show blood-brain barrier crossing ability and tumor targeted aggregation effect.
  • Figure 42 shows that the third degradation tool (NTZ-NP) in Example 8 obtains blood-brain barrier crossing ability and tumor targeting ability through simple self-assembly, and is loaded with fluorescent dye DIR for tracking, and is traced through a small animal imager Detect brain aggregation and statistical graph of DIR fluorescence.
  • NTZ-NP third degradation tool
  • the left picture of Figure 42 shows that when glioma tumor-bearing mice were injected with the nanodegradation tool Ang-NTZ-NP containing 20% Ang-2 arm, the DIR dye signal in the brain accumulated significantly.
  • the right picture shows three biological replicate experiments. Statistics, the picture on the right shows that the aggregation level of Ang-NTZ-NP in the brain is significantly increased, with a significant difference.
  • nanoparticles have drug-loading capabilities and can also be modified and self-assembled simply through plug-and-play to obtain blood-brain barrier crossing capabilities and tumor targeting capabilities.
  • This example uses the optional third degradation tool obtained in Example 3, in which the POI recognition group is NTZ, the connecting arm is DSPE-PEG, and the nanoparticle core is PLGA.
  • NTZ-NP can obtain the blood-brain barrier crossing ability and tumor targeting ability through a simple "plug and play", and perform protein degradation and tumor suppression of the target protein EGFR on an orthotopic tumor model. Investigate therapeutic potential in vivo.
  • the preparation method is as described in Experiment 3 of this example.
  • When self-assembling the NTZ-PEG-DSPE arm to the PLGA core add 20% Angiopep2-PEG-DSPE with blood-brain barrier crossing ability, self-assemble with PLGA together, and then purify. Concentrate and obtain Ang/NTZ-NP.
  • mice On the 15th day after human glioma transplantation into 6-8 week old female nude mice, 10 mg/kg was administered intravenously. Equimolar amounts of control and equal volumes of solvent control were also injected simultaneously into the tail vein. A total of 5 doses were administered once every two days. After completing the administration cycle, the mice were euthanized the next day, and the brain tissue was removed and embedded in paraffin, and sections were subjected to immunohistochemical staining to study the protein of tumor proliferation markers PCNA and NTZ's target protein EGFR. degradation effect.
  • Figure 43 of the present disclosure shows the immunohistochemical detection of EGFR and proliferation marker PCNA after brain tumor dissection after the third degradation tool (NTZ-NP) of Example 8 was administered to the glioma orthotopic animal model through the assembly method of Figure 42 Result graph.
  • the results showed that EGFR and PCNA in the solvent control PBS group and NTZ-NP group were stronger than those in the ANG/NTZ-NP group. It shows that this nano-protein degradation tool has good application potential (the arrow marks the representative positive signal.
  • the scale bar is 50 ⁇ m).
  • the degradation tool in Example 9 was prepared, including coupling a POI recognition group to a lipid hybrid substance, in which the POI recognition group was the monoclonal antibody drug Nitrozumab (NTZ).
  • the POI recognition group was the monoclonal antibody drug Nitrozumab (NTZ).
  • Lipid hybrid substances are PEG (polyethylene glycol) liposomes, that is, liposomes (liposomes) contain PEG exposed on the surface of lipid hybrid substances, and their PEG ends have active reaction sites NHS (N-hydroxyl) Succinimide) is used for amino coupling with the POI recognition group NTZ antibody.
  • the product structure obtained in this example is NTZ-PEGlipo.
  • Pretreatment Replace the original antibody buffer with PBS.
  • the EGFR monoclonal antibody Nimotuzumab (NTZ) which has been used in clinical treatment, is used.
  • nimotuzumab solution centrifuge it at 4000 ⁇ g for 2 minutes with a 3kDa ultrafiltration tube, and then concentrate the antibody, and then dilute it with PBS; after repeated concentration and dilution, the buffer is replaced with PBS, and after dilution, it is passed through the NanoDrop one C instrument IgG mode determines protein concentration with absorbance at A280, and the results are verified with the BCA protein quantification kit.
  • HSPC hydrogenated soybean lecithin
  • cholesterol cholesterol
  • cholesterolesterol cholesterolesterol
  • DSPE disearoylphosphatidylethanolamine
  • PEG Polyethylene glycol
  • HSPC and CHO were dissolved in chloroform, then evaporated, concentrated and dried to form a film.
  • the coupling reaction was started in an ice-water mixture environment and protected by nitrogen.
  • the lipid hybrid solution was then added to the antibody PBS solution stirred at 800 rpm (revolutions per minute); the stirred reaction was then incubated at 4°C on a 20 rpm rotator for 24 hours.
  • the reaction mixture was then concentrated three times by ultrafiltration using a 50 kDa centrifugal ultrafiltration tube, and then diluted to 100 ⁇ L with PBS. Protein concentration was determined by absorbance at A280 by NanoDrop one C instrument in IgG mode, and its results were verified by BCA protein quantification kit.
  • Figure 47 is a diagram of the synthesis process of NTZ-PEGlipo.
  • Figure 48 shows the results of protein electrophoresis to detect EGFR protein expression after MDA-MB-231 (M231) human breast cancer tumor cells were treated with 500 nM NTZ-PEGlipo for 24 hours. The results show that NTZ-PEGlipo can effectively degrade the target protein EGFR.
  • the degradation tool in Example 10 was prepared, that is, the POI recognition group was coupled to the amphipathic lipid connecting arm, and then self-assembled with the lipid hybrid substance.
  • the POI recognition group in Example 10a and Example 10b is: monoclonal antibody drug nitrozine (NTZ); the connecting arm is NHS-PEG-DSPE with an NHS group.
  • the lipid hybrid material is composed of a membrane skeleton and cholesterol (CHO, cholesterolol).
  • the membrane skeleton is hydrogenated soybean lecithin (HSPC), and the degradation tool it consists of is NTZ-lipo1.
  • the membrane skeleton is 2 Stearoylphosphatidylcholine (DSPC), whose degradation tool is NTZ-lipo2.
  • the POI recognition group is: INE, Inetetamab, a therapeutic monoclonal antibody for HER2.
  • HER2 is a tumor proliferation signal receptor and marker, and the connecting arm is NHS-PEG-DSPE with an NHS group.
  • the lipid hybrid material is composed of HSPC membrane skeleton and CHO, and its degradation tool is INE-lipo.
  • the POI recognition group is: small molecule CDK4 inhibitor Palbociclib (Palb), the connecting arm is NHS-PEG-DSPE with NHS group, Palbociclib is coupled through the reaction of amino group and NHS, and the lipid hybrid substance It is composed of HSPC membrane skeleton and CHO, and its degradation tool is Palb-lipo.
  • the POI recognition group is an antibody.
  • the POI recognition group in Example 10a and Example 10b is: monoclonal antibody drug nitrozine (NTZ).
  • the POI recognition group is: monoclonal antibody drug Initor (INE).
  • Pretreatment Replace the original antibody buffer with phosphate buffered saline PBS.
  • the NTZ and INE monoclonal antibody solutions were taken, centrifuged at 4000 ⁇ g for 2 minutes using a 3kDa ultrafiltration tube, and then the antibodies were concentrated, and then diluted with PBS; after repeated concentration and dilution three times, the main component of the buffer was replaced with PBS , after the antibody solution is diluted, the protein concentration is determined by the absorbance at A280 in the mode of the NanoDrop one C instrument, and the protein concentration is determined by the BCA protein quantification reagent. box to verify its results.
  • Non-directed site coupling includes amino coupling, carboxyl coupling, and bridged thiol coupling.
  • Site-directed coupling such as click chemical reaction, selenium bond coupling, serine coupling, cysteine coupling, non-natural amino acid coupling, enzyme catalyzed post-coupling, and sugar site coupling.
  • HSPC, DSPC, and CHO are dissolved in chloroform, and then evaporated, concentrated, and dried to form a film.
  • NTZ-lipo1, NTZ-lipo2, and INE-lipo After the solvent chloroform of the aforementioned lipid hybrid substance solution is completely evaporated, add 1 mL of DSPE-PEG-antibody PBS solution, and then ultrasonicate at room temperature for 3 minutes at 100W. The resulting solution was then passed through 800nm, 400nm and 200nm filters respectively. Subsequently, for further purification, the reaction mixture was ultrafiltrated and concentrated three times using a 50 kDa centrifugal ultrafiltration tube, and then diluted to 100 ⁇ L with PBS. Protein concentration was determined by absorbance at A280 by NanoDrop one C instrument in IgG mode, and its results were verified by BCA protein quantification kit.
  • the POI recognition group is a small molecule.
  • the POI recognition group is Palbociclib small molecule drug or ⁇ -amyloid probe AV-45.
  • Palbociclib is a clinically commercial CDK4 inhibitor, and the conjugate of its lipid hybrid substance in the embodiment is Palb-lipo.
  • CDK4 is a cyclin-dependent kinase that is widely involved in cell senescence and tumorigenesis.
  • Palbociclib reacts with NHS-PEG (2kDa)-DSPE by reacting the amino group on Palbociclib with the NHS group at the end of DSPE-PEG (molar ratio 1.2:1). Under nitrogen protection at room temperature, the reaction was stirred for 24 hours to obtain a Palb-PEG-DSPE reaction mixture. Palb-PEG-DSPE was dialyzed for 24 hours and lyophilized, and the concentration was determined by the standard curve of the UV absorption peak.
  • the preparation of lipid hybrid material is the same as Experiment 1 of this example.
  • AV-45 is a ⁇ -amyloid probe, a classic marker of Alzheimer's disease, and its lipid hybrid substance conjugate is AV45-lipo.
  • ⁇ -amyloid is a potential pathogenic protein of Alzheimer's disease. It is an extracellular protein. Its precursor is expressed on the cell membrane. After being cleaved, it is secreted extracellularly to form oligomers ( ⁇ -amyloid 1-42oligomer) and fibers. Among them, oligomers are considered to be the most toxic. As the accumulation of extracellular protein ⁇ -amyloid oligomers brings pathological risks such as neuroinflammation, isotope-labeled AV-45 is a clinically commercial ⁇ -amyloid probe. This example uses an isotope-free AV-45 derivative, AV-45-SH. AV-45 has blue autofluorescence, which facilitates cell tracking, and its sulfhydryl SH group is used to mix with Mal (maleimide)- PEG-DSPE conjugation.
  • Preparation method of AV45-PEG-DSPE Combine AV45-SH with thiol group and Mal-PEG (molecular weight 2kDa)-DSPE, that is, dissolve AV45-SH and Mal-PEG-DSPE in DMSO, the molar ratio is 1.2:1, and protect from light Nitrogen protection in 37°C water bath for 24h. Subsequently dialyzed and lyophilized. Concentrations were determined by passing the standard curve of UV absorption peaks.
  • lipid hybrid material is the same as Experiment 1 of this example.
  • Preparation of AV45-lipo After the solvent chloroform of the aforementioned lipid hybrid substance solution is completely evaporated, add 1 mL of AV45-PEG-DSPE in PBS solution, and then ultrasonicate at room temperature for 3 minutes at 100W. The resulting solution was then passed through 800nm, 400nm and 200nm filters respectively. Determine the concentration through the standard curve of the UV absorption peak, and conduct cell experiments based on the AV45 molar concentration.
  • Figure 49 is a schematic diagram of the synthesis process.
  • Figure 50 shows the results of protein electrophoresis to detect EGFR protein expression after M231 human breast cancer tumor cells were treated with different concentrations of NTZ-lipo1 for 24 hours. The results show that NTZ-lipo1 can effectively degrade the target protein EGFR.
  • Figure 51 shows the protein electrophoresis results of the degradation effect of the target protein EGFR on M231 cells treated with NTZ-lipo1 composed of different HSPC and CHO ratios for 24 hours. The results show that different compositions have protein degradation effects, while the membrane skeleton HSPC and auxiliary The ratio of lipids to cholesterol has an impact on protein degradation.
  • Figure 52 shows the results of protein electrophoresis to detect EGFR protein expression after M231 human breast cancer tumor cells were treated with 500nM NTZ-lipo2 for 24 hours. The results show that NTZ-lipo2 can effectively degrade the target protein EGFR.
  • Figure 53 shows the results of protein electrophoresis to detect HER2 protein expression after M231 human breast cancer tumor cells were treated with 500nM INE-lipo for 24 hours. The results show that INE-lipo can effectively degrade the target protein.
  • Figure 54 shows the hydrogen nuclear magnetic resonance spectrum ( 1 H NMR) of Palb-PEG-DSPE after the lipid linking arm used in Palb-lipo is coupled with the POI recognition group. The results show that the connection is successful.
  • Figure 65 shows the hydrogen nuclear magnetic resonance spectrum of AV45-PEG-DSPE after the lipid linking arm used in AV45-lipo was coupled with the POI recognition group. The results show that the connection was successful.
  • Figure 56 shows the results of protein electrophoresis to detect CDK4 protein expression after M231 human breast cancer tumor cells were treated with different concentrations of Palb-lipo for 24 hours. The results showed that Palb-lipo can effectively degrade the target protein.
  • Figure 57 is a schematic structural diagram of AV45-lipo and the structure of AV45-SH.
  • Figure 58 shows confocal microscopy imaging of human glial cells after being treated with different concentrations of AV45-lipo for 24 hours to detect the effect of ⁇ -amyloid (A ⁇ ) being hijacked into the cells.
  • the oligomers prepared from ⁇ -amyloid 1-42 polypeptide labeled with 10 ⁇ M FITC green dye were incubated with 10 ⁇ M AV45-lipo for 24 hours, and then the processed substances that did not enter the cells were washed away with PBS, and confocal microscopy imaging was performed to detect ⁇ -The green fluorescence of amyloid 1-42 oligomers.
  • the lysosomal dye LysoTracker is used to label lysosomes.
  • the scale bar is 10 ⁇ m.
  • the degradation tool in Example 11 was prepared, that is, the POI recognition group was coupled to the amphipathic lipid linking arm, and then self-assembled with the lipid hybrid substance.
  • the POI recognition group is: monoclonal antibody drug Nitrozine (NTZ); the connecting arm is NHS-PEG-DSPE with NHS group or NHS-PEG-DMG (1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol, polyethylene glycol terminal modified N-hydroxysuccinimide).
  • the lipid hybrid substance is composed of the membrane skeleton HSPC, cholesterol, and the cationic lipid DOTAP (trimethyl-2,3-dioleoyloxypropylammonium bromide).
  • Cationic lipid DOTAP is a representative cationic lipid. Cationic lipids help lipid hybrid substances form Lipid nanoparticles (LNP), which are used to encapsulate negatively charged nucleic acid drugs.
  • LNP Lipid nanoparticles
  • the degradation tool is NTZ-LNP1.
  • NTZ-LNP1 carries nonsense sequence empty small interfering RNA (siRNA)
  • siRNA small interfering RNA
  • Example 10 The method used in Example 10 was adopted for its pretreatment, connection of POI recognition group and connecting arm, and purification.
  • HSPC and CHO were dissolved in chloroform, then evaporated, concentrated and dried to form a film.
  • the reaction mixture was then ultrafiltrated and concentrated three times using a 50kDa centrifugal ultrafiltration tube, and then diluted to 100 ⁇ L with PBS. Protein concentration was determined by absorbance at A280 by NanoDrop one C instrument in IgG mode, and its results were verified by BCA protein quantification kit.
  • Figure 59 is a schematic structural diagram of NTZ-LNP1, NTZ-LNP1s, and NTZ-LNP2.
  • Figure 60 shows the particle size distribution of the lipid hybrid material detected by dynamic light scattering (DLS).
  • Figure 61 shows the results of protein electrophoresis to detect EGFR protein expression after M231 human breast cancer tumor cells were treated with 500 nM NTZ-LNP1 for 24 hours. Lipo1 in Example 10 was also used as a control. The results showed that NTZ-LNP1 could effectively degrade the target protein EGFR.
  • Figure 62 shows the results of protein electrophoresis to detect EGFR protein expression after M231 human breast cancer tumor cells were treated with NTZ-LNP1s for 24 hours.
  • Figure 63 shows the results of protein electrophoresis to detect EGFR protein expression after M231 human breast cancer tumor cells were treated with NTZ-LNP2 for 24 hours. The results show that NTZ-LNP2 can effectively degrade the target protein EGFR.
  • the degradation tool in Example 12 was prepared, that is, the POI recognition group was coupled to the amphipathic lipid linking arm, and then self-assembled with the lipid hybrid substance.
  • the POI recognition group is: monoclonal antibody drug nitrozine (NTZ); the connecting arm is NHS-PEG-DSPE with an NHS group.
  • Lipid hybrid substances are composed of exosomes. Exosomes are extracellular vesicles secreted by eukaryotes such as animal or plant cells. They are formed spontaneously and can carry out intercellular communication. They usually contain proteins and a small amount of nucleic acids. Exosomes have high biocompatibility and Drug-loading capacity, its surface is a phospholipid membrane structure.
  • Example 10 The method used in Example 10 was adopted for its pretreatment, connection of POI recognition group and connecting arm, and purification.
  • Exosomes were isolated, purified and identified from DC2.4 cells according to standard procedures. The exosomes were coupled to the connecting arms by ultrasound and membrane extrusion. That is, add 1 mL of DSPE-PEG-antibody PBS solution to the exosome PBS solution.
  • the connecting arm and lipid hybrid substance are combined by extrusion or ultrasonic method.
  • the reaction mixture was then ultrafiltrated and concentrated three times using a 50kDa centrifugal ultrafiltration tube, and then diluted to 100 ⁇ L with PBS. Protein concentration was determined by absorbance at A280 by NanoDrop one C instrument in IgG mode, and its results were verified by BCA protein quantification kit.
  • Figure 64 shows the particle size and average dispersity coefficient PDI (Polymer dispersity index) of the lipid hybrid substance detected by DLS. The smaller the PDI, the particle size is approximately uniform, and the result indicates that the particle size is uniform.
  • Figure 65 shows the results of protein electrophoresis to detect EGFR protein expression after M231 human breast cancer tumor cells were treated with different concentrations of NTZ-exo for 24 hours. The results show that NTZ-exo can effectively degrade the target protein EGFR.
  • PDI Polymer dispersity index
  • the degradation tool in Example 13 was prepared, that is, the lipid hybrid substance was coated on the surface of the nanoparticle, and its POI recognition group was coupled to the amphiphilic lipid linking arm. Subsequently, it self-assembles with the lipid hybrid material, and the POI recognition group is exposed to the outside.
  • the POI recognition group is: EGFR-targeting monoclonal antibody drug nitrozine (NTZ) or cetuximab (CTX); the connecting arm is NHS-PEG-DSPE with an NHS group.
  • NTZ EGFR-targeting monoclonal antibody drug nitrozine
  • CTX cetuximab
  • the connecting arm is NHS-PEG-DSPE with an NHS group.
  • the degradation tool is NTZ-lipoP.
  • Polylactic acid-co-glycolic acid is a representative polymer nanoparticle. It is hydrophobic, has high biocompatibility, stable properties and can be degraded by organisms, and is widely used in medical treatment.
  • the lipid hybrid material is composed of mouse red blood cell membrane (RBCm) and wrapped with acetalized dextran (Dextran). When the POI recognition group is composed of CTX, the degradation tool is CTX-RBCmD. Dextran is a representative hydrophilic biodegradable nanoparticle.
  • Red blood cell membrane is a representative of cell membrane and organelle membrane. It is a lipid hybrid substance that is easy to separate and extract.
  • Example 10 The method used in Example 10 was adopted for its pretreatment, connection of POI recognition group and connecting arm, and purification.
  • Coating PLGA Dissolve PLGA (15kDa) in DMF (10 ⁇ g/ ⁇ L), add 2 ⁇ L every 10 seconds into 1mL PBS solution, stir at 700rpm for 3h, wait After DMF evaporates, each 0.01 ⁇ mol of PLGA is wrapped with 1 mg of LIPO (referring to 1 mg of HSPC + CHO in total). Dissolve appropriate amounts of HSPC and CHO in chloroform, evaporate and concentrate, then resuspend with DSPE-PEG-NTZ PBS solution, 100W Ultrasound for 3 minutes. Incubate for 30 minutes on a shaking table at 37°C, ultrasonicate at 100W for 5 minutes at room temperature, and pass through 800, 400, and 200nm membranes. The mixture of the two was then mixed with PLGA's PBS solution, ultrasonicated in an ice bath at 100W for 2 minutes, passed through 800, 400, and 200nm membranes, and concentrated by ultrafiltration. IgG concentration was measured.
  • CTX-RBCmD The synthesis method of CTX-PEG-DSPE is as in Example 10.
  • Figure 66 is a schematic diagram of the NTZ-lipoP structure.
  • Table 6 shows the DLS particle size statistics and PDI statistical results of NTZ-lipoP and lipo-PLGA without NTZ-PEG-DSPE connection. The results show that the particle size is uniform, and the hydrated particle size increases slightly after connecting NTZ-PEG-DSPE.
  • Figure 67 shows the results of protein electrophoresis to detect EGFR protein expression after M231 human breast cancer tumor cells were treated with different concentrations of NTZ-lipoP for 24 hours. The results showed that it can effectively degrade the target protein EGFR.
  • Figure 68 is a schematic structural diagram of CTX-RBCmD.
  • Figure 69 is a graph of DLS particle size distribution results.
  • Figure 70 shows the results of protein electrophoresis to detect EGFR protein expression after M231 human breast cancer tumor cells were treated with 500nM CTX-RBCmD for 24 hours. The results showed that it can effectively degrade the target protein EGFR.
  • the POI recognition group is modified on the surface of the nanoparticle, or the POI recognition group is connected to the connecting arm, or the POI recognition group is modified on the connecting arm and connected to the surface of the nanoparticle through the connecting arm.
  • NP nanoparticles
  • the POI recognition group is assembled, Exposed to the outside of nanoparticles or connecting arms, where nanoparticles (NP, Nanoparticles) can penetrate cells independently of specially designed receptor-ligand matching pairs, and can be coupled to small molecules, peptides, nucleic acid aptamers, POI recognition groups such as antibodies specifically carry out protein hijacking and targeted degradation, thereby realizing the assembly of nanoparticle-based TPD tools (TPD-NP).
  • TPD-NP nanoparticle-based TPD tools
  • This convenient nanoparticle-based TPD tool has easy access to drug loading and tissue-specific targeting capabilities, making possible combined drug and protein degradation therapy as well as translational/precision medicine.
  • the invention of TPD-NP and the exploration of its mechanism have greatly expanded the scope of TPD tools and provided basic knowledge for the fields of TPD and nanodelivery, and can in principle degrade a variety of human disease-related extracellular/membrane-associated/ intracellular proteins.
  • the disclosed nano-protein degradation tool has a flexible structure, is easy to transform, and can obtain the ability to carry drugs, target, and cross biological barriers.
  • the disclosed nano-protein degradation tool is universal, the target can be replaced at will, all three components can be replaced, and the application scenarios are wide. All components of the disclosed nano-protein degradation tool can be clinically approved materials, with high in vivo application potential and translational value.
  • the disclosed nano-protein degradation tool does not need to be synthesized from scratch and is a ready-to-use platform, which greatly reduces the complexity of development and production. sex and difficulty.
  • TPD-NP can degrade extracellular/intracellular/membrane proteins.
  • extracellular/membrane protein degradation tools such as LYTAC
  • TPD-NP does not require additional design of structures to assist the hijacked protein in being endocytosed.
  • the nano-protein degradation tool of the present disclosure does not require a special structure to guide protein degradation.
  • Nanoparticles can be loaded with drugs, can be designed for controlled release, can be designed into synergistic treatment materials such as photothermal magnetism, etc., and can be used for imaging, contrast, and protein degradation for synergistic treatment and integration of diagnosis and treatment.
  • the body fluid stability of NP can reduce drug loss and improve drug potential.
  • the present disclosure achieves target protein degradation based on lipid hybrid substances by modifying the POI recognition group on the surface of the lipid hybrid substance, and exposing the POI recognition group to the outside of the lipid hybrid substance after assembly. It greatly reduces the difficulty of synthesizing protein degradation tools.
  • a large number of compounds, peptides, antibodies and nucleic acid aptamers that have binding ability to target proteins can be upgraded into protein degradation drugs, and then It can play new functions in traditional liposome and lipid nanoparticle (LNP) related fields, such as mRNA vaccines, nucleic acid delivery carriers, and drug delivery, thereby enabling the development of combination therapies in scientific research and industrial applications.
  • LNP liposome and lipid nanoparticle
  • the plug-and-play combination method adopted in this disclosure is extremely convenient and convenient, and the structure can be flexibly assembled as needed.
  • the assembly materials can be completely derived from clinically acceptable materials, and creatively endow the nanocarrier with protein delivery properties other than Degradation function also broadens the scope of biotech drugs.
  • lipid nanoparticles lipid hybrid substances
  • This invention greatly expands the current application scope of lipid nanoparticles and It provides fundamental knowledge for the fields of TPD and nanodelivery, and can in principle degrade a variety of human disease-related extracellular/membrane-associated/intracellular proteins in vivo.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nanotechnology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)

Abstract

本公开提供了一种纳米蛋白降解工具、应用及其制备方法。其中，所述纳米蛋白降解工具包括：第一降解工具、第二降解工具和第三降解工具中的一种或多种组合；其中，第一降解工具由POI识别基团与连接臂连接构成；第二降解工具由POI识别基团与纳米颗粒连接构成；第三降解工具由POI识别基团通过连接臂与纳米颗粒连接构成。本公开实现了基于纳米粒子的TPD-NP的组装，使得构建蛋白降解工具的合成难度极大降低，通过"即插即用"模式，实现海量与靶蛋白具有结合力的化合物、多肽、抗体、核酸适配体能够升级成为蛋白降解类药物，进而进行转化医学/精准医学的疗法开发。TPD-NP的发明和其机制的探索，大大扩展了目前TPD工具的范围，并为TPD和纳米递送领域提供了基础知识，并且原则上可以在体内降解多种人类疾病相关的细胞外/膜相关/细胞内蛋白。本公开还提供了一种基于脂质的蛋白降解工具、应用及其制备方法。其中，所述基于脂质的蛋白降解工具包括：POI识别基团，以及与所述POI识别基团连接的脂质杂化物质。通过POI识别基团修饰于脂质杂化物质的表面，并且，POI识别基团组装后暴露于脂质杂化物质的外部，实现了基于脂质杂化物质的靶蛋白降解，使得构建蛋白降解工具的合成难度极大降低，通过"即插即用"模式，使得海量与靶蛋白具有结合力的化合物、多肽、抗体和核酸适配体等能够升级成为蛋白降解类药物，进而在脂质体、LNP相关领域中发挥新的功能，从而在科研和工业应用中实现联合疗法开发。

Description

一种纳米蛋白降解工具、应用及其制备方法以及一种基于脂质的蛋白降解工具、应用及其制备方法
相关申请的交叉引用
本公开要求于2022年7月29日提交中国国家知识产权局的申请号为202210906564.6、名称为“一种纳米蛋白降解工具、应用及其制备方法”的中国专利申请以及于2022年7月29日提交中国国家知识产权局的申请号为202210911853.5、名称为“一种基于脂质的蛋白降解工具、应用及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开属于靶向药物技术领域，尤其涉及一种纳米蛋白降解工具、应用及其制备方法以及一种基于脂质的蛋白降解工具、应用及其制备方法。
背景技术
靶向蛋白降解工具(TPD，targeted protein degradation)是特异性地将感兴趣的蛋白质(POI，Protein of interest)劫持到细胞内蛋白质回收站，以实现靶向蛋白质降解。TPD已成为生物医学研究和制药行业的强大工具。
与传统的酶抑制/拮抗小分子药物相比，蛋白水解靶向嵌合体(PROTAC)作为最具代表性的第一代TPD的代表成员，能够靶向降解传统难以成药的靶点，但是靶点仅限胞内蛋白。胞外蛋白和膜蛋白在疾病发生发展中扮演重要角色，基因编码的总蛋白中约40％是非胞内蛋白。为了将降解目标蛋白的范围扩大到细胞质之外，前期有研究团队开发了靶向溶酶体的嵌合体(LYTAC)，通过阳离子磷酸寡甘露糖尾连接抗体/多肽，借助甘露糖-6-磷酸受体发挥内吞进入溶酶体降解。随后，该团队利用肝细胞富含去唾液酸糖蛋白受体(ASGPR)的特性，将LYTAC磷酸甘露糖更换为三半乳糖结构，进而升级为肝脏特异性LYTAC，LYTAC方法虽然具有应用潜力，但合成方法复杂。LYTAC是受体依赖的，需要在LYTAC结构上特殊设计，帮助蛋白复合物进入细胞，同时针对不同的细胞靶向，需要将尾巴进行更换，因此在设计和合成方面工作繁琐而困难。
目前公认的TPD工具的成功开发，需要设计①合适的靶蛋白识别基团②劫持蛋白进入细胞和胞内运输的受体-配体匹配对，③启动适当的蛋白质降解机制，④从成药角度需要针对细胞类型特异性设计靶向能力和生物屏障穿透能力。然而，包括PROTAC、LYTAC等类似的主流TPD工具，在针对任何新POI开发工具时，都需要费力的个案设计，其中不乏需要从头合成化合物并大量筛选的过程，才能用于不同的疾病和细胞类型。
综上，现有的靶向蛋白降解工具存在设计和合成方法复杂、需要针对每一个案疾病和细胞类型独立设计，灵活性差，不便于改造，不便于体内靶向递送，不具有血脑屏障等生物屏障穿越能力的缺陷，为针对患者的靶向治疗方式带来了巨大的不便。
发明内容
为解决上述问题，本公开提供了一种纳米蛋白降解工具，包括：第一降解工具、第二降解工具和第三降解工具中的一种或多种组合；
其中，所述第一降解工具由POI识别基团与连接臂连接构成；所述第二降解工具由所述POI识别基团与纳米颗粒连接构成；所述第三降解工具由所述POI识别基团通过所述连接臂与所述纳米颗粒连接构成；
所述POI识别基团包括能与POI特异性结合的抗体、蛋白、多肽、核酸适配体或小分子。
可选地，所述第一降解工具中，所述POI识别基团与所述连接臂构成一组连接单元；所述第一降解工具是由包括多组设于核心处的所述连接臂以及与所述连接臂连接的、设于外围的所述POI识别基团的所述连接单元所组成的具有多层结构的所述纳米蛋白降解工具；
所述第二降解工具是由包括设于核心处的所述纳米颗粒以及与所述纳米颗粒连接的、设于外围的多个所述POI识别基团的连接单元所组成的具有多层结构的所述纳米蛋白降解工具；
所述第三降解工具中，所述POI识别基团通过所述连接臂与所述纳米颗粒连接；所述第三降解工具是由包括设于核心处的所述纳米颗粒，与所述纳米颗粒连接的、设于中间层的多组所述连接臂以及设于外围的、与所述连接臂连接的所述POI识别基团的连接单元所组成的具有多层结构的所述纳米蛋白降解工具。
可选地，所述连接臂包括亲水聚合物、疏水聚合物和两亲性聚合物。
可选地，所述连接臂的分子量为0-1000kDa。
可选地，所述两亲性聚合物包括：两亲性嵌段聚合物，所述亲水聚合物与疏水小分子组成的两亲性聚合物，以及所述疏水聚合物与亲水小分子组成的两亲性聚合物。
可选地，所述两亲性聚合物为链状或支化分子结构的聚合物，其至少存在一个具有亲水性的分子端，以及一个具有疏水性的分子端。
可选地，所述两亲性聚合物为直链分子结构，其一端为亲水性的分子端，另一端为具有疏水性的分子端。
可选地，所述纳米颗粒包括表面单一纳米颗粒和杂化纳米颗粒；
所述表面单一纳米颗粒包括亲水性颗粒、疏水性颗粒和无机纳米颗粒；
所述杂化纳米颗粒为通过杂化物质对所述表面单一纳米颗粒改性后的杂化纳米颗粒；
其中，所述杂化物质为改性膜；所述改性膜包括细胞膜、外泌体、油膜、水凝胶和脂质体。
可选地，所述杂化纳米颗粒为通过所述改性膜包覆于所述表面单一纳米颗粒外表面构成的颗粒，以便于所述表面单一纳米颗粒在改性成为所述杂化纳米颗粒后，能与所述亲水聚合物的臂、疏水聚合物的臂、或所述POI识别基团相连接。
所述纳米颗粒的粒径为5-1000nm；
可选地，所述第三降解工具中，所述纳米颗粒为疏水性颗粒、亲水性颗粒或所述表面单一纳米颗粒外包覆有所述改 性膜，且所述连接臂为两亲性聚合物、亲水聚合物或者疏水聚合物时，所述连接臂与所述纳米颗粒的连接方式包括：所述连接臂通过非共价键合于所述纳米颗粒上，或者通过所述连接臂上修饰的活性基团共价键合于所述纳米颗粒上。
可选地，所述POI识别基团中的抗体为治疗性单克隆抗体、多特异性抗体、纳米抗体及前述抗体衍生物或抗体偶联药物；
所述多肽为具有POI特异性结合能力的多肽；
所述小分子为具有POI特异性结合能力的小分子化合物。
此外，为解决上述问题，本公开还提供了一种如上述纳米蛋白降解工具在制备治疗和预防蛋白异常积累疾病的药物、疫苗、递送载体中的应用；其中，所述蛋白异常积累疾病包括肿瘤、免疫系统疾病、炎症和病原体感染、神经退行性疾病、血液系统疾病和代谢类疾病。
此外，为解决上述问题，本公开还提供了一种治疗和预防蛋白异常积累疾病的方法，包括向受试者施用治疗有效量的上述纳米蛋白降解工具，其中，所述蛋白异常积累疾病包括肿瘤、免疫系统疾病、炎症和病原体感染、神经退行性疾病、血液系统疾病和代谢类疾病。
此外，为解决上述问题，本公开还提供了一种如上述纳米蛋白降解工具在制备针对蛋白异常积累疾病的检测产品和/或试剂盒中的应用；其中，所述蛋白异常积累疾病包括肿瘤、免疫系统疾病、炎症和病原体感染、神经退行性疾病、血液系统疾病和代谢类疾病。
此外，为解决上述问题，本公开还提供了一种如上述纳米蛋白降解工具的制备方法，包括：
所述第一降解工具和所述第二降解工具的制备包括：将所述POI识别基团通过非共价键合到所述纳米颗粒或连接臂上，或者，将所述POI识别基团基于活性基团通过共价键合到所述纳米颗粒或连接臂上，从而构成所述纳米蛋白降解工具；
所述第三降解工具的制备包括：
先将所述POI识别基团与所述连接臂偶联，构成偶联中间体；然后将所述偶联中间体连接到所述颗粒核心部上，构成所述第三降解工具；或者
先构建由连接臂作为外层且由纳米颗粒作为核心的纳米复合结构；再将所述POI识别基团与所述纳米颗粒的复合结构偶联，构成所述纳米蛋白降解工具。
本公开提供了一种纳米蛋白降解工具、应用及其制备方法，其中所述纳米蛋白降解工具包括：第一降解工具、第二降解工具和第三降解工具中的一种或多种组合；其中，所述第一降解工具由POI识别基团与连接臂连接构成；所述第二降解工具由所述POI识别基团与纳米颗粒连接构成；所述第三降解工具由所述POI识别基团通过所述连接臂与所述纳米颗粒连接构成；所述POI识别基团包括能与POI特异性结合的抗体、蛋白、多肽、核酸适配体或小分子。
本公开还提供了一种基于脂质的蛋白降解工具，包括：
POI识别基团，以及与所述POI识别基团连接的脂质杂化物质；
其中，所述POI识别基团包括能与POI特异性结合的抗体、蛋白、多肽、核酸适配体或小分子；
所述脂质杂化物质包括脂质体、外泌体、细胞膜以及LNP。
可选地，在所述POI识别基团与所述脂质杂化物质偶联时，所述基于脂质的蛋白降解工具是由包括处于核心的所述脂质杂化物质与设于外围的所述POI识别基团组成的用于蛋白降解的纳米颗粒。
可选地，所述基于脂质的蛋白降解工具还包括，在所述POI识别基团与所述脂质杂化物质之间设有连接构件的所述基于脂质的蛋白降解工具。
可选地，所述连接构件的分子量为0-1000kDa；
所述连接构件为聚合物连接臂和脂质连接臂中的一种。
可选地，所述聚合物连接臂包括亲水聚合物、疏水聚合物和两亲性聚合物。
可选地，所述脂质连接臂为两亲性的脂质连接臂。
可选地，在所述POI识别基团通过所述连接构件与所述脂质杂化物质偶联时，所述POI识别基团与所述连接构件构成一组连接单元；所述基于脂质的蛋白降解工具是由包括设于核心处的所述脂质杂化物质，设于中间层的、与所述脂质杂化物质连接的多组所述连接构件以及设于外围的、与所述连接构件连接的所述POI识别基团的多组连接单元所组成的具有多层结构的所述基于脂质的蛋白降解工具。
可选地，所述脂质连接臂中至少包括两端，一端为能与所述脂质杂化物质相连接的亲脂末端，另一端为亲水末端。
可选地，亲脂末端为脂质分子。
可选地，所述两亲性聚合物为链状或支化分子结构的聚合物，其至少存在一个具有亲水性的分子端，以及一个具有疏水性的分子端。
可选地，所述两亲性聚合物为直链分子结构，其一端为亲水性的分子端，另一端为具有疏水性的分子端。
可选地，还包括纳米颗粒，其中，所述纳米颗粒被所述脂质杂化物质包覆于所述基于脂质的蛋白降解工具的核心处；
所述纳米颗粒包括亲水性颗粒、疏水性颗粒和无机纳米颗粒。
可选地，所述纳米颗粒的粒径为5-1000nm。
此外，为解决上述问题，本公开还提供了一种如上述基于脂质的蛋白降解工具，在制备治疗和预防有害蛋白异常积累疾病的药物及递送系统中的应用；其中，所述有害蛋白异常积累疾病包括肿瘤、免疫系统疾病、神经退行性疾病、血液系统疾病和代谢类疾病。
此外，为解决上述问题，本公开还提供了一种治疗和预防蛋白异常积累疾病的方法，包括向受试者施用治疗有效量的上述基于脂质的蛋白降解工具，其中，所述蛋白异常积累疾病包括肿瘤、免疫系统疾病、炎症和病原体感染、神经退行性疾病、血液系统疾病和代谢类疾病。
此外，为解决上述问题，本公开还提供了一种如上述基于脂质的蛋白降解工具，在制备针对有害蛋白异常积累疾病 的检测产品和/或试剂盒中的应用；其中，所述有害蛋白异常积累疾病包括肿瘤、免疫系统疾病、神经退行性疾病、血液系统疾病和代谢类疾病。
此外，为解决上述问题，本公开还提供了一种如上述基于脂质的蛋白降解工具的制备方法，在所述基于脂质的蛋白降解工具为所述POI识别基团与所述脂质杂化物质偶联的蛋白降解工具时，其制备方法为：将所述POI识别基团通过非共价键合到所述脂质杂化物质上；或者，将所述POI识别基团基于偶联基团通过共价键合到所述脂质杂化物质上，从而构成所述基于脂质的蛋白降解工具。
可选地，所述基于脂质的蛋白降解工具还包括所述POI识别基团通过连接构件与所述脂质杂化物质连接的蛋白降解工具；
其中，在所述基于脂质的蛋白降解工具为所述POI识别基团通过所述连接构件与所述脂质杂化物质连接的蛋白降解工具时，其制备方法为：
先将所述POI识别基团与所述连接构件偶联，构成偶联中间体；然后将所述偶联中间体连接到所述脂质杂化物质上，构成所述基于脂质的蛋白降解工具；或者
先构建由所述连接构件作为外层且由所述脂质杂化物质作为核心的纳米复合结构；再将所述POI识别基团与所述纳米复合结构偶联，构成所述基于脂质的蛋白降解工具。
本公开提供了一种基于脂质的蛋白降解工具、应用及其制备方法，其中所述基于脂质的蛋白降解工具包括：POI识别基团，以及与所述POI识别基团连接的脂质杂化物质；其中，所述POI识别基团包括能与POI特异性结合的抗体、蛋白、多肽、核酸适配体或小分子；所述脂质杂化物质包括脂质体、外泌体、细胞膜以及LNP。
附图说明
为了更清楚地说明本公开实施方式的技术方案，下面将对实施方式中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施方式，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本公开纳米蛋白降解工具的第一降解工具的结构示意图；
图2为本公开纳米蛋白降解工具的第二降解工具的结构示意图；
图3为本公开纳米蛋白降解工具的第三降解工具的结构示意图；
图4为本公开实施例1中第一降解工具合成示意图；
图5为本公开实施例1中第一降解工具考马斯亮蓝染色结果；
图6为本公开实施例1中第一降解工具蛋白降解效果的蛋白电泳结果；
图7为本公开实施例2中第二降解工具合成示意图；
图8为本公开实施例2中第二降解工具JQ1-NP纳米颗粒所用JQ1-NH2结构和JQ1-NP纳米颗粒动态光散射结果；
图9为本公开实施例2中第二降解工具JQ1-NP蛋白电泳结果；
图10为本公开实施例2中第二降解工具NTZ-NP2蛋白电泳结果；
图11为本公开实施例3中第三降解工具合成示意图；
图12为本公开实施例3中第三降解工具结构形态示意图；
图13为本公开实施例3中第三降解工具动态光散射和透射电镜结果；
图14为本公开实施例3中第三降解工具(NTZ-NP)在人乳腺癌M231细胞、人宫颈癌HeLa细胞、人脑胶质瘤U87细胞蛋白降解效果的蛋白电泳结果；
图15为在M231细胞经第三降解工具处理后的免疫荧光染色结果；
图16为M231细胞经第三降解工具处理后的固定视野活细胞时间序列拍摄绿色荧光标记的EGFR蛋白降解结果；
图17为HepG2细胞经第三降解工具处理后的CCK8检测细胞活力结果；
图18为本公开实施例3中第三降解工具的EGFR蛋白表达；其中，a为有效降解浓度探索；b为处理后蛋白恢复情况探索；
图19为本公开实施例3中第三降解工具的EGFR蛋白表达，探索包含POI识别基团和连接臂构成的偶联中间体与不含POI识别基团的空白连接臂之间不同的比例对降解效果的影响；
图20为本公开实施例4中第三降解工具(NTZ-AuNP)的结构示意图；
图21为本公开实施例4中第三降解工具(NTZ-AuNP)的蛋白降解效果电泳结果；
图22为本公开实施例5中第三降解工具(ACE2-NP)中POI识别基团与连接臂连接产物(ACE2-PEG-DSPE)的核磁共振氢谱(1H NMR)；
图23为本公开实施例5中第三降解工具(CD13-NP)中POI识别基团与连接臂连接产物(CD13-PEG-DSPE)的核磁共振氢谱(1H NMR)；
图24为本公开实施例5中第三降解工具(ACE2-NP)处理被GFP标记ACE2的293细胞后的流式细胞术检测GFP结果图；
图25为本公开实施例5中第三降解工具(CD13-NP)处理人肝癌细胞HepG2后的蛋白电泳结果图；
图26为本公开实施例5中第三降解工具(AB-NP)处理人胶质细胞后，共聚焦显微镜检测FITC绿色染料标记的β-amyloid 1-42寡聚体的细胞内吞的实验结果；
图27为本公开实施例6中第三降解工具第三降解工具(Palb-NP)中POI识别基团与连接臂连接产物(Palb-PEG-DSPE)的核磁共振氢谱(1H NMR)；
图28为本公开实施例6中第三降解工具(Palb-NP)的蛋白降解效果的蛋白电泳图；
图29为本公开实施例6中第三降解工具处理M231细胞后结晶紫染色克隆形成实验结果；
图30为本公开实施例6中第三降解工具(AV45-NP)结构示意图；
图31为本公开实施例6中第三降解工具(AV45-NP)中POI识别基团与连接臂连接产物(AV45-PEG-DSPE)的核磁共 振氢谱；
图32为本公开实施例6中第三降解工具(AV45-NP)处理人胶质细胞后，共聚焦显微镜检测FITC绿色染料标记的β-amyloid 1-42寡聚体的细胞内吞的实验结果；
图33为本公开实施例7中第三降解工具(CTX-NP)的蛋白降解效果的蛋白电泳图；
图34为本公开实施例7中第三降解工具(PTZ-NP)的蛋白降解效果的共聚焦显微镜图；
图35为本公开实施例7中第三降解工具(ATZ-NP)的蛋白降解效果的蛋白电泳图；
图36为本公开实施例7中第三降解工具(CRLZ-NP)的蛋白降解效果的蛋白电泳图；
图37为本公开实施例7中第三降解工具(INE/NTZ-NP)的蛋白降解效果的蛋白电泳图；
图38和图39为本公开实施例8中第三降解工具(NTZ-NP)的动物体内实验；其中，图38中a为NTZ-NP处理M231细胞裸鼠皮下瘤模型期间裸鼠肿瘤体积；b为NTZ-NP处理结束解剖分离肿瘤进行蛋白电泳检测EGFR表达并统计；c为NTZ-NP处理期间裸鼠体重变化；
图39为NTZ-NP处理结束后分离肿瘤组织进行免疫荧光染色检测EGFR结果；
图40为NTZ-NP处理结束后分离肿瘤组织进行免疫荧光染色检测凋亡标志物结果；
图41为本公开实施例8中第三降解工具(NTZ-NP)单次给药后白鼠的血液肝功能肾功能指标检测情况；
图42为本公开实施例8中第三降解工具(NTZ-NP)通过简单的自组装获得血脑屏障穿越能力和肿瘤靶向能力，并装载了荧光染料DIR进行示踪，通过小动物成像仪检测DIR荧光的脑部聚集情况和统计图；
图43为本公开实施例8第三降解工具(NTZ-NP)通过图42的组装方式给与胶质瘤原位动物模型后脑肿瘤解剖后的免疫组化检测EGFR和增殖标志物PCNA结果图；
图44为本公开POI识别基团与脂质杂化物质偶联的结构示意图；
图45为本公开含有连接构件的POI识别基团与脂质杂化物质偶联的结构示意图；
图46为本公开含有连接构件-脂质连接臂的POI识别基团与脂质杂化物质连接的结构示意图；
图47为本公开实施例9中NTZ-PEGlipo合成结构示意图；
图48为本公开实施例9中NTZ-PEGlipo在M231细胞中对靶蛋白EGFR降解效果的蛋白电泳结果；
图49为本公开实施例10中NTZ-lipo1、NTZ-lipo2、INE-lipo、Palb-lipo、AV45-lipo合成示意图；
图50为本公开实施例10中不同浓度NTZ-lipo1在M231细胞中对靶蛋白EGFR降解效果的蛋白电泳结果；
图51为本公开实施例10中不同脂质杂化物质组成比例的NTZ-lipo1在M231细胞中对靶蛋白EGFR降解效果的蛋白电泳结果；
图52为本公开实施例10中NTZ-lipo2在M231细胞中对靶蛋白EGFR降解效果的蛋白电泳结果；
图53为本公开实施例10中INE-lipo在M231细胞中对靶蛋白HER2降解效果的蛋白电泳结果；
图54为本公开实施例10中Palb-lipo所用脂质连接臂与POI识别基团偶联后产物的核磁共振氢谱(1H NMR)结果；
图55为本公开实施例10中AV45-lipo所用脂质连接臂与POI识别基团偶联后产物的核磁共振氢谱(1H NMR)结果；
图56为本公开实施例10中不同浓度Palb-lipo在M231细胞中对靶蛋白CDK4降解效果的蛋白电泳结果；
图57为本公开实施例10中AV45-lipo结构示意图；
图58为本公开实施例10中AV45-lipo与β-amyloid(Aβ)寡聚体共孵育在人胶质细胞培养基中，随后PBS洗涤后共聚焦显微镜检测细胞溶酶体标记、荧光染料FITC标记的Aβ和AV45自发荧光的结果图；
图59为本公开实施例11中NTZ-LNP1、NTZ-LNP1s、NTZ-LNP2的结构示意图；
图60为本公开实施例11中NTZ-LNP1、NTZ-LNP1s、NTZ-LNP2动态光散射(DLS)粒径分布图结果；
图61为本公开实施例11中NTZ-LNP1以及实施例10中NTZ-lipo1在M231细胞中对靶蛋白EGFR降解效果的蛋白电泳结果；
图62为本公开实施例11中NTZ-LNP1s在M231细胞中对靶蛋白EGFR降解效果的蛋白电泳结果；
图63为本公开实施例11中NTZ-LNP2在M231细胞中对靶蛋白EGFR降解效果的蛋白电泳结果；
图64为本公开实施例12中NTZ-exo的DLS粒径分布和平均分散系数PDI图；
图65为本公开实施例12中NTZ-exo在M231细胞中对靶蛋白EGFR降解效果的蛋白电泳结果；
图66为本公开实施例13中NTZ-lipoP结构示意图；
图67为本公开实施例13中不同浓度NTZ-lipoP在M231细胞中对靶蛋白EGFR降解效果的蛋白电泳结果；
图68为本公开实施例13中CTX-RBCmD结构示意图；
图69为本公开实施例13中CTX-RBCmD的DLS粒径分布结果图；
图70为本公开实施例13中CTX-RBCmD在M231细胞中对靶蛋白EGFR降解效果的蛋白电泳结果。
附图标记：
100，基于脂质的蛋白降解工具；1，POI识别基团；2，脂质杂化物质中的脂质体、外泌体或细胞膜；3，脂质杂化物质中的LNP；4，脂质连接臂；5，聚合物连接臂；6，偶联基团；7，纳米颗粒。
本公开目的的实现、功能特点及优点将结合实施例，参照附图做进一步说明。
具体实施方式
下面将结合实施例对本公开的技术方案进行清楚、完整地描述，显然，所描述的实施例是本公开一部分实施例，而不是全部的实施例。基于本公开中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本公开保护的范围。
除非在下文中另有定义，本公开具体实施方式中所用的所有技术术语和科学术语的含义意图与本领域技术人员通常所理解的相同。虽然相信以下术语对于本领域技术人员很好理解，但仍然阐述以下定义以更好地解释本公开。
如本公开中所使用，术语“包括”、“包含”、“具有”、“含有”或“涉及”为包含性的(inclusive)或开放式的，且不排除其它 未列举的元素或方法步骤。术语“由…组成”被认为是术语“包含”的可选的实施方案。如果在下文中某一组被定义为包含至少一定数目的实施方案，这也应被理解为揭示了一个可选地仅由这些实施方案组成的组。
在提及单数形式名词时使用的不定冠词或定冠词例如“一个”或“一种”，“所述”，包括该名词的复数形式。
本公开中的术语“大约”表示本领域技术人员能够理解的仍可保证论及特征的技术效果的准确度区间。该术语通常表示偏离指示数值的±10％，可选地为±5％。
此外，说明书和权利要求书中的术语第一、第二、第三、(a)、(b)、(c)以及诸如此类，是用于区分相似的元素，不是描述顺序或时间次序必须的。应理解，如此应用的术语在适当的环境下可互换，并且本公开描述的实施方案能以不同于本公开描述或举例说明的其它顺序实施。
以下仅仅是为了帮助理解本公开而提供。这些定义不应被理解为具有小于本领域技术人员所理解的范围。
下面结合具体实施例的方式对本公开的技术方案做进一步的详细说明，但并不构成对本公开的任何限制，任何人在本公开权利要求范围内所做的有限次的修改，仍在本公开的权利要求范围之内。
本公开提供了一种纳米蛋白降解工具，包括：
第一降解工具(其结构参考图1)、第二降解工具(其结构参考图2)和第三降解工具(其结构参考图3)中的一种或多种组合；
其中，所述第一降解工具由POI识别基团与连接臂连接构成；所述第二降解工具由所述POI识别基团与纳米颗粒连接构成；所述第三降解工具由所述POI识别基团通过所述连接臂与所述纳米颗粒连接构成；
所述POI识别基团包括能与POI特异性结合的抗体、蛋白、多肽、核酸适配体或小分子。
所述POI识别基团是能和POI特异性结合的抗体、蛋白、多肽、核酸适配体或小分子。
所述纳米颗粒，即为纳米微粒或纳米粒子，其可以为实心的颗粒结构、空心结构或多孔颗粒结构，在颗粒结构中成分或分子结构可以为单一成分，也可以为多组分复合物。其中，纳米颗粒可以为具有生物相容性的纳米颗粒。
所述连接臂为可以有利于改善水溶性和稳定性的结构和物质，起到与POI识别基团连接，或者起到POI识别基团和纳米颗粒之间的桥接作用，使其组成一个整体结构。
所述纳米蛋白降解工具包括三种表现形式(结构形态)，分别为第一降解工具、第二降解工具和第三降解工具。结构形态，由如下表格中展示：
表1、纳米蛋白降解工具(TPD-NP)的三种结构形式
所述POI识别基团包括：能与POI特异性结合的抗体、蛋白、多肽、核酸适配体或小分子。
其中，抗体可以包括治疗性单克隆抗体、多特异性抗体、纳米抗体及前述抗体衍生物或抗体偶联药物；
蛋白可以为：具有POI特异性结合能力的蛋白；
多肽为具有POI特异性结合能力的多肽；包括ACE2、CD13、β-Amyloid的binding多肽；
核酸适配体为具有POI特异性结合能力的核酸适配体；
小分子为具有POI特异性结合能力的小分子化合物，包括CDK4/6蛋白抑制剂帕博西尼(Palbociclib)的衍生物，BRD4蛋白抑制剂JQ1的衍生物,β-Amyloid蛋白探针AV-45的衍生物，匹兹堡化合物PiB的衍生物，Tau蛋白探针GTP1、PBB3的衍生物。所述抗体包括：CTX、NTZ、PTZ、CRLZ、INE、ATZ、AND和MTX。其中，CTX(Cetuximab)西妥昔单抗是最经典的人鼠嵌合EGFR蛋白单抗，已获全球100多个国家/地区批准，用于治疗RAS野生型转移性结直肠癌,局部晚期及复发和转移头颈部鳞状细胞癌。NTZ(Nimotuzumab)尼妥珠单抗是中国地区最经典的EGFR蛋白单抗，用于治疗多种由于EGFR突变造成的肿瘤。PTZ(pertuzumab)帕妥珠单抗是由罗氏在2012年研发上市的靶向HER2的单抗药物,和曲妥珠单抗与HER2蛋白的结合位点不同。CRLZ(Camrelizumab)PD-1抑制剂卡瑞利珠单抗，是经典免疫治疗单抗，用于晚期肺癌、肝癌、食管癌和霍奇金淋巴瘤的治疗。INE(Inetetamab)伊尼妥单抗是HER2单抗，目前用于HER2阳性乳腺癌的治疗。ATZ(Atezolizumab)是一种PD-L1人源化单克隆抗体，是经典的肿瘤免疫治疗靶点。阿杜那单抗ADN(aducanumab)，是一种用于阿尔兹海默症的单克隆抗体，能够结合β-amyloid蛋白。MTX(Miltuximab)是抗磷脂酰肌醇蛋白聚糖1的单克隆抗体，是新兴的肿瘤治疗靶点。
本公开通过将POI识别基团修饰于纳米颗粒的表面，或POI识别基团连接于连接臂，或POI识别基团修饰于连接臂通过连接臂连接于纳米颗粒表面，POI识别基团组装后暴露于纳米颗粒或连接臂的外部，其中，纳米颗粒能够不依赖特殊设计的受体-配体匹配对进行细胞渗透，并且可以偶联小分子、多肽、核酸适配体、抗体等POI识别基团，特异性地进行蛋白质劫持和靶向降解，从而实现针对于基于纳米颗粒(NP,Nanoparticle)的TPD工具(TPD-NP)的组装。这种具有简便载药和组织特异性的靶向纳米蛋白降解工具，使得药物和蛋白降解工具治疗以及转化/精准医学成为可能。TPD-NP的发明和其机制的探索，大大扩展了TPD工具的范围，并为TPD和纳米递送领域提供了基础知识，并且原则上可以在体内降解多种人类疾病相关的细胞外/膜相关/细胞内蛋白。
可选地，在纳米蛋白降解工具中，三种降解工具其整体在构建后为如下表所示的空间结构形态：
表2、纳米蛋白降解工具(TPD-NP)的三种空间结构形态

所述纳米蛋白降解工具，由于不同的连接方式，组成的TPD-NP的空间结构有所不同。
其中，第一降解工具中，由于其为POI识别基团与连接臂构成一组连接单元，则整体为多层结构，内部为核心，外部为外围，将多个连接单元置入该多层结构中，其形态即为连接臂一端在核心处，与连接臂连接的POI识别基团处于外围，构成一个由内至外的多层结构的颗粒形态的TPD-NP。
其中，第二降解工具中，由于其为POI识别基团与纳米颗粒连接，则其空间结构形态同样是由多层机构构成，内部为核心，外部为外围，内部核心为单一的纳米颗粒，而与之连接的多个POI识别基团处于外围，构成一个内外双层结构的颗粒形态的第二降解工具。
其中，第三降解工具中，由于其连接方式为POI识别基团通过连接臂与纳米颗粒连接，即“POI识别基团-连接臂-纳米颗粒”。在第三降解工具中，整体为多层结构，分为三层，内部为核心，中部为中间层，外部是外围。基于连接方式，内部核心为一个纳米颗粒，则与该纳米颗粒连接的多个连接臂处于中间层，在外围则是与这些连接臂连接的POI识别基团，构成一个三层结构的颗粒形态的第三降解工具。
可选地，连接臂包括亲水聚合物、疏水聚合物和两亲性聚合物；
根据其水溶性的性质，所述连接臂可以分为3种不同的连接臂，包括：1、亲水聚合物，2、疏水聚合物，3、两亲性聚合物。
所述亲水聚合物可以包括但不限于：聚乙二醇(PEG)、聚环氧乙烷(PEO)、聚(乙二醇)甲基丙烯酸酯(POEG)、聚2-甲基丙烯酰氧基乙基磷酰(PMPC)、聚羧酸甜菜碱(PCB)、葡聚糖、透明质酸、壳聚糖、β-环糊精、超支化聚缩水甘油醚(HPG)、聚N-(2-羟丙基)甲基丙烯酰胺(PHPMA)、聚甲基丙烯酸羟乙酯(PHEMA)、聚丙烯酰胺(PAM)、聚乙烯吡咯烷酮(PVP)、聚丙烯酸(PAA)、聚马来酸酐(HPMA)、聚季胺盐及其药学上可接受的聚合物盐、聚乙烯亚胺(PEI)、聚甲基丙烯酸N,N-二甲基氨基乙酯(PDMAEMA)、聚赖氨酸(PLL)、聚谷氨酸(PGu)及聚天冬氨酸(PAsp)等各类亲水性聚氨基酸，及上述聚合物的衍生物及其药学上可接受的盐。
所述疏水聚合物可以包括但不限于：聚乳酸-羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚己内酯(PCL)、聚碳酸酯(PMC)及其衍生物、乙交酯/丙交酯/己内酯/碳酸酯各类组合及组分的共聚物、聚氨酯(PU)、聚醚醚酮(PEEK)、聚甲基丙烯酸甲酯(PMMA)、聚乙烯醇(PVA)、聚乙烯(PE)、聚苯丙氨酸等各类疏水聚氨基酸及上述聚合物的衍生物及其药学上可接受的盐。
所述两亲性聚合物可以包括但不限于：PEG-PLGA、PEG-PCL、PEG-PLA、PEG-PMC，及上述亲水聚合物和疏水聚合物组合的各种两亲性嵌段聚合物及衍生物，以及上述亲水聚合物与疏水分子(如脂质)组成的各种两亲性聚合物及衍生物。上述脂质分子可以包括但不限于：脂肪酸类(fatty acids)，甘油脂类(glycerolipids)，甘油磷脂类(glycerophospholipids)，鞘脂类(sphingolipids)，固醇脂类(sterol lipids)，孕烯醇酮脂类(prenol lipids)，糖脂类(saccharolipids)，多聚乙烯类(polyketides),阳离子脂质(cationic lipids)和可电离脂质(ionizable lipids)及其衍生物。
可选地，所述连接臂的分子量为0-1000kDa；
可选地，所述两亲性聚合物包括：两亲性嵌段聚合物和所述亲水聚合物与疏水小分子组成的两亲性聚合物，以及所述疏水聚合物与亲水小分子组成的两亲性聚合物。
所述两亲性聚合物包括3种，其结构形式分别为如下表格中所示：
表3、两亲性聚合物的三种结构形式
其中，两亲性嵌段聚合物是指直链或分支状，在单一聚合物分子中存在两种或两种以上结构不同的链段，可根据需要合成具有特定化学结构、分子量的共聚物。具有两亲性的嵌段聚合物指在溶液中可自组装成特定的超分子有序聚集体(胶束或囊泡)。两亲嵌段聚合物指在水中溶解后可自发形成由亲水性外壳和亲脂性内核组成的高分子胶束，或亲水性外壳和亲脂性中间层及亲水空腔组成的高分子囊泡。其余两种分别为，亲水聚合物与疏水小分子组成的两亲性聚合物，以及疏水聚合物与亲水小分子组成的两亲性聚合物。在一个可选的技术方案中，两亲性聚合物为链状或支化分子结构的聚合物，其至少存在一个具有亲水性的分子端，以及一个具有疏水性的分子端。
所述两亲性聚合物为链状分子结构的聚合物，可以为多个直链相连接而组成的分子结构的聚合物，其分子结构中至少有一个亲水的分子端和一个疏水的分子端，从而使该两亲聚合物具有两亲性。
可选地，所述两亲性聚合物为直链分子结构，其一端为亲水性的分子端，另一端为具有疏水性的分子端。
上述，该两亲聚合物为直链结构，该直链的分子结构包括两个分子端，其一为亲水分子端，另一端为疏水分子端。
需要说明的是，由于连接臂的功能可以为：对所述纳米颗粒进行水溶性改性或改善POI识别基团的空间位阻。因此，当需要增加纳米颗粒水溶性及改善POI识别基团空间位阻时，就需要使用连接臂对纳米颗粒进行连接。
如果纳米颗粒为有机高分子纳米颗粒中的脂溶性聚合物、水溶性聚合物或无机纳米颗粒，则需要在纳米蛋白降解工具中采用连接臂改善纳米颗粒的稳定性，并桥接于纳米颗粒和POI识别基团之间。
而当纳米颗粒为两亲聚合物时，其同时具有亲水性和疏水性的两亲性，因此并不需要提高或改善其亲水性，此时不需要利用连接臂的功能，直接用纳米颗粒与POI识别基团连接。
可选地，纳米颗粒包括表面单一纳米颗粒和杂化纳米颗粒；
所述表面单一纳米颗粒包括亲水性颗粒、疏水性颗粒和无机纳米颗粒。
所述无机纳米颗粒包括金纳米颗粒、碳纳米颗粒、硅纳米颗粒、氧化铁纳米颗粒、磷酸钙纳米颗粒、硫酸钡和碘化物造影剂、氮化铝纳米颗粒、氧化铝纳米颗粒、氧化钛纳米颗粒、铝铁合金颗粒、钛铁合金颗粒，均为具有稳定且低生物毒性的材料。
所述单一纳米颗粒可以为具有生物相容性的材料。可选地，所述单一纳米颗粒为生物可降解材料。
所述亲水性颗粒可以为树枝状聚合物、超支化聚合物、以及前述的亲水聚合物及其衍生物组成的各种纳米凝胶、以及纳米白蛋白。
所述疏水性颗粒可以为聚乳酸-羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚己内酯(PCL)、聚碳酸酯(PMC)及其衍生物、乙交酯/丙交酯/己内酯/碳酸酯的共聚物、聚氨酯(PU)、聚醚醚酮(PEEK)、聚甲基丙烯酸甲酯(PMMA)、聚乙烯醇(PVA)、聚苯乙烯(PS)、聚乙烯(PE)、聚苯丙氨酸等各类疏水聚氨基酸及其衍生物制备的纳米颗粒。所述杂化纳米颗粒为通过杂化物质对所述表面单一纳米颗粒改性后的杂化纳米颗粒。
其中，所述杂化物质为改性膜；所述改性膜包括细胞膜、外泌体、油膜、水凝胶和脂质体。
所述杂化物质可以为改性膜。通过改性膜对于纳米颗粒进行包覆，使其具有一定的水溶性的改变。所述改性膜可以包括细胞膜、外泌体、油膜、水凝胶和脂质体。
可选地，所述杂化纳米颗粒为通过所述改性膜包覆于所述表面单一纳米颗粒外表面构成的颗粒，以便于所述表面单一纳米颗粒在改性成为所述杂化纳米颗粒后，能与所述亲水聚合物、或疏水聚合物的臂相连接，或者与所述两亲性聚合物臂相连接，或者与所述POI识别基团相连接。
所述纳米颗粒的粒径为5-1000nm；
所述第三降解工具中，所述纳米颗粒为疏水性颗粒、亲水性颗粒或所述表面单一纳米颗粒外包覆有所述改性膜的杂化纳米颗粒。当所述连接臂为两亲性聚合物、亲水聚合物或者疏水聚合物时，所述连接臂与所述纳米颗粒的连接方式包括：所述连接臂通过非共价键合于所述纳米颗粒上，或者通过所述连接臂上修饰的活性基团共价键合于所述纳米颗粒上；
可选地，所述POI识别基团中的抗体为治疗性单克隆抗体、多特异性抗体、纳米抗体及前述抗体衍生物或抗体偶联药物；
所述多肽为具有POI特异性结合能力的多肽；包括ACE2、CD13、β-Amyloid的binding多肽；
核酸适配体为具有POI特异性结合能力的核酸适配体；
所述小分子为具有POI特异性结合能力的小分子化合物，包括CDK4/6蛋白抑制剂帕博西尼(Palbociclib)的衍生物，BRD4蛋白抑制剂JQ1的衍生物,β-Amyloid蛋白探针AV-45的衍生物，匹兹堡化合物PiB的衍生物，Tau蛋白探针GTP1、PBB3的衍生物。
所述抗体包括：CTX、NTZ、PTZ、CRLZ、INE、ATZ、AND和MTX。其中，CTX(Cetuximab)西妥昔单抗是最经典的人鼠嵌合EGFR蛋白单抗，已获全球100多个国家/地区批准，用于治疗RAS野生型转移性结直肠癌,局部晚期及复发和转移头颈部鳞状细胞癌。NTZ(Nimotuzumab)尼妥珠单抗是中国地区最经典的EGFR蛋白单抗，用于治疗多种由于EGFR突变造成的肿瘤。PTZ(Pertuzumab)帕妥珠单抗是由罗氏在2012年研发上市的靶向HER2的单抗药物,和曲妥珠单抗与HER2蛋白的结合位点不同。CRLZ(Camrelizumab)PD-1抑制剂卡瑞利珠单抗，是经典免疫治疗单抗，用于晚期肺癌、肝癌、食管癌和霍奇金淋巴瘤的治疗。INE(Inetetamab)伊尼妥单抗是HER2单抗，目前用于HER2阳性乳腺癌的治疗。ATZ(Atezolizumab)是一种PD-L1人源化单克隆抗体，是经典的肿瘤免疫治疗靶点。阿杜那单抗ADN(aducanumab)，是一种用于阿尔兹海默症的单克隆抗体，能够结合β-amyloid蛋白。MTX(Miltuximab)是抗磷脂酰肌醇蛋白聚糖1的单克隆抗体，是新兴的肿瘤治疗靶点。
此外，本公开还提供了一种纳米蛋白降解工具，在制备治疗和预防蛋白异常积累疾病的药物、疫苗、递送载体中的应用；其中，所述蛋白异常积累疾病包括肿瘤、免疫系统疾病、神经退行性疾病、血液系统疾病和代谢类疾病。
此外，本公开还提供了一种纳米蛋白降解工具，在制备针对蛋白异常积累疾病的检测产品和/或试剂盒中的应用；其中，所述蛋白异常积累疾病包括肿瘤、免疫系统疾病、炎症和病原体感染、神经退行性疾病、血液系统疾病和代谢类疾病。
在本公开中此类蛋白降解工具被用于多种病理相关蛋白降解，并且在动物肿瘤模型中实验原位肿瘤病理相关蛋白降解。
应用范围包括但不限于肿瘤、免疫系统疾病、炎症和病原体感染、神经退行性疾病、血液系统疾病和代谢相关疾病等涉及有害蛋白异常积累的疾病中的药物开发和诊断，以及体外检测和生物医药研究中蛋白质降解工具的开发，以及蛋白互作研究中的试剂盒开发。
此外，本公开还提供了一种纳米蛋白降解工具的制备方法，其特征在于，包括：
(1)针对于所述第一或第二降解工具的制备方法1：将所述POI识别基团通过非共价键合到所述纳米颗粒或连接臂上；
(2)针对于所述第一或第二降解工具的制备方法2：将所述POI识别基团基于活性基团通过共价键合到所述纳米颗粒或连接臂上，从而构成所述纳米蛋白降解工具；
(3)针对于所述第三降解工具的制备方法1：先将所述POI识别基团与所述连接臂偶联，构成偶联中间体；然后将所述偶联中间体连接到所述颗粒核心部上，构成所述第三降解工具；
(4)针对于所述第三降解工具的制备方法2：先构建由连接臂作为外层且由纳米颗粒作为核心的纳米复合结构；再将所述POI识别基团与所述纳米颗粒的复合结构偶联，构成所述纳米蛋白降解工具。
上述，在制备、构建或组装纳米蛋白降解工具时，可以包括但不限于上述3种方式，可以针对于第一、第二和第三 降解工具选择对应的方法进行制备对应的蛋白降解工具。
上述，(1)和(2)中，分别为两种制备第一或第二降解工具的方法，可以根据纳米颗粒的性质选择，(3)和(4)中为两种制备第三降解工具的方法，其区别在于，连接顺序不同，(3)中为先将POI识别基团于连接臂偶联，再与颗粒核心部连接，(4)中为先将连接臂与纳米颗粒连接，构成复合结构后再与POI识别基团偶联。
参考图44，本公开还提供了一种基于脂质的蛋白降解工具，包括：
POI识别基团，以及与所述POI识别基团连接的脂质杂化物质；
其中，所述POI识别基团包括能与POI特异性结合的抗体、蛋白、多肽、核酸适配体或小分子；
所述脂质杂化物质包括脂质体、外泌体、细胞膜以及LNP(lipid nanoparticle)。
所述POI识别基团是能和POI特异性结合的抗体、蛋白、多肽或小分子。
所述脂质杂化物质，包括脂质体(liposome)、外泌体、细胞膜以及LNP。其中，LNP，即为脂质纳米颗粒(lipid nanoparticle)。
需要说明的是，脂质类纳米颗粒，主要用于药物体内递送，从传统脂质体(liposome)到脂质纳米颗粒(LNP，lipid nanoparticle)，已经在小分子药物递送、核酸类药物递送、纳米疫苗等领域被广泛应用于医疗。其中COVID-19的mRNA疫苗多由LNP组成。
脂质类纳米颗粒具有成本低廉便于制备的优点，其组成规律也被研究较为清楚，用于医疗和药物开发的脂质类纳米颗粒拥有一套较为规律的组成，最基本的为膜骨架和胆固醇辅料。临床在用的脂质类纳米颗粒，根据需要也可以添加其他组分，可以包括但不限于如下组分：
1、膜骨架，组成脂质双分子层膜的主要成分。
2、辅助辅料：(1)胆固醇(cholesterol)，调节膜流动性，提高粒子稳定性，(2)辅助磷脂，维持脂质体微观形态，使溶酶体膜不稳定。
3、PEG化脂质，减少粒子在体内与血浆蛋白的结合，延长体循环时间。
4、阳离子脂质，高效包载核酸药物，提供正电荷，体内转染，pH敏感(可电离型)。
5、稳定剂，具有冻干保护作用，维持脂质体在冻干过程中的结构稳定。
所述脂质杂化物质包括脂质体、外泌体、细胞膜、LNP、LPP以及脂质纳米乳。其中，脂质体(liposome)，也称为经典脂质体，其形态为空泡状，其主要用于载疏水药物。由于脂质体作为一种两亲性膜，疏水空泡里可以载疏水药物，亲水空泡中，可以载亲水药物。而LNP为脂质纳米颗粒，其内部含有阳离子脂质，目前mRNA疫苗和一些核酸类疫苗就是用的LNP，因核酸带负电，阳离子脂质带正电，可以能够有效提高包载效率，LNP也被用于CRISPR基因编辑元件的递送。而传统脂质体lipo载核酸效率低，一般是载疏水、亲水小分子药物。LPP因包含了聚合物核心获得了脂质和聚合物分别的特性，具有更好的载药兼容性和稳定性。
此外，LNP和LPP还具有便于被修饰和改造的优点，通过两亲性PEG化脂质和类似的结构，极易容易对脂质体、LNP进行修饰，PEG化脂质可以通过PEG末端连接配体(ligands)用于和受体结合，可能促进药物对靶器官的递送，也被称为主动靶向的脂质类纳米载体。
脂质杂化物质便于载药，所述药物包括亲疏水性小分子药物、核酸类药物、蛋白类药物、基因编辑载体等生物技术类药物均可以被有效装载和递送。但其应用仍然集中在药物递送，其结构本身具有的生理、病理性质，仍需被进一步探索，更多的治疗潜力和可能性值得被探索。
总之，本公开通过将POI识别基团修饰于脂质杂化物质的表面，并且，POI识别基团组装后暴露于脂质杂化物质的外部，实现了基于脂质杂化物质的靶蛋白降解，使得构建蛋白降解工具的合成难度极大降低，通过“即插即用”模式，使得海量与靶蛋白具有结合力的化合物、多肽、抗体和核酸适配体等能够升级成为蛋白降解类药物，进而在传统脂质体(Liposome)、脂质纳米颗粒(LNP)相关领域，如mRNA疫苗、核酸递送载体、药物递送中发挥新的功能，从而实现在科研和工业应用中进行联合疗法开发。
此外，配体靶向的脂质类纳米颗粒(脂质杂化物质)作为蛋白降解工具的应用和其机制的探索尚属空白，该发明大大扩展了目前脂质类纳米颗粒的应用范围，并为TPD和纳米递送领域提供了基础知识，并且原则上可以在体内降解多种人类疾病相关的细胞外/膜相关/细胞内蛋白。
可选地，在所述POI识别基团与所述脂质杂化物质偶联时，所述基于脂质的蛋白降解工具是由所述脂质杂化物质处于核心，以所述POI识别基团设于外围组成的用于蛋白降解的纳米颗粒。
上述基于脂质的蛋白降解工具的结构中，整体可以为颗粒状的结构，包括核心处和外围，核心部分可以为一个脂质杂化物质，外围为多个与脂质杂化物质连接的POI识别基团，即在脂质杂化物质的外表面有一层与之连接的众多POI识别基团，从而构成了整体的基于脂质的蛋白降解工具。
此外，根据脂质杂化物质的形态，其原则上，如果为经典脂质体，其形态为空泡状。
可选地，参考图45，所述基于脂质的蛋白降解工具还可包括一种，在所述POI识别基团与所述脂质杂化物质之间设有连接构件的所述基于脂质的蛋白降解工具。
可选地，所述连接构件的分子量为0-1000kDa；
所述连接构件为聚合物连接臂和脂质连接臂中的一种；其中，含有脂质连接臂的基于脂质的蛋白降解工具结构如图46所示。
可选地，所述聚合物连接臂包括亲水聚合物、疏水聚合物和两亲性聚合物。
可选地，所述脂质连接臂为两亲性的脂质连接臂。
本公开提供了另外一种基于脂质的蛋白降解工具的结构，与基本结构相类似的，在脂质杂化物质和POI识别基团之间，还设有连接构件。构成了“脂质杂化物质-连接构件-POI识别基团”的依次连接的组合，其中连接构件上设有能与POI识别基团连接的偶联基团，从而能够实现两者的组合。
可选地，在所述POI识别基团通过所述连接构件与所述脂质杂化物质偶联时，所述POI识别基团与所述连接构件构成一组连接单元；所述基于脂质的蛋白降解工具是由包括设于核心处的所述脂质杂化物质，设于中间层的、与所述脂质杂化物质连接的多组所述连接构件以及设于外围的、与所述连接构件连接的所述POI识别基团的多组连接单元所组成的具有多层结构的所述基于脂质的蛋白降解工具。
此种情况下该基于脂质的蛋白降解工具在空间上的结构，其核心处为一个脂质杂化物质(核心)，其表层连接有多个连接构件(中间层)，并且每个连接构件均连接有一脂质杂化物质(外围)，构成了内外三层的颗粒状的结构，其整体理论上可以为球形，也可以为其他形状。
可选地，所述连接构件的分子量为0-1000kDa。
所述连接构件为聚合物连接臂和脂质连接臂中的一种。
可选地，所述聚合物连接臂包括亲水聚合物、疏水聚合物和两亲性聚合物。
表4、聚合物连接臂的三种结构形式
可选地，所述脂质连接臂为两亲性的脂质连接臂。
根据连接构件的特征，其分为两大类，包括：聚合物连接臂和脂质连接臂，可以根据物质的合成需要、水溶性需要而选择任意一种，并且，质连接臂为两亲性的脂质连接臂。
所述亲水聚合物可以包括但不限于：聚乙二醇(PEG)、聚环氧乙烷(PEO)、聚(乙二醇)甲基丙烯酸酯(POEG)、聚2-甲基丙烯酰氧基乙基磷酰(PMPC)、聚羧酸甜菜碱(PCB)、葡聚糖、透明质酸、壳聚糖、β-环糊精、超支化聚缩水甘油醚(HPG)、聚N-(2-羟丙基)甲基丙烯酰胺(PHPMA)、聚甲基丙烯酸羟乙酯(PHEMA)、聚丙烯酰胺(PAM)、聚乙烯吡咯烷酮(PVP)、聚丙烯酸(PAA)、聚马来酸酐(HPMA)、聚季胺盐及其药学上可接受的聚合物盐、聚乙烯亚胺(PEI)、聚甲基丙烯酸N,N-二甲基氨基乙酯(PDMAEMA)、聚赖氨酸(PLL)、聚谷氨酸(PGu)及聚天冬氨酸(PAsp)等各类亲水性聚氨基酸及上述聚合物的衍生物及其药学上可接受的盐。
疏水聚合物可以包括：聚乳酸-羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚己内酯(PCL)、聚碳酸酯(PMC)及其衍生物、乙交酯/丙交酯/己内酯/碳酸酯各类组合及组分的共聚物、聚氨酯(PU)、聚醚醚酮(PEEK)、聚甲基丙烯酸甲酯(PMMA)、聚乙烯醇(PVA)、聚乙烯(PE)、聚苯丙氨酸等各类疏水聚氨基酸及上述聚合物的衍生物及其药学上可接受的盐。
两亲性聚合物包括：PEG-PLGA、PEG-PCL、PEG-PLA、PEG-PMC，及上述亲水聚合物和疏水聚合物组合的各种两亲性嵌段聚合物及衍生物，以及上述亲水聚合物与疏水分子(如脂质)组成的各种两亲性聚合物及衍生物。
可选地，所述脂质连接臂中至少包括两端，一端为能与所述脂质杂化物质相连接的亲脂末端，另一端为亲水末端；
可选地，所述亲脂末端为脂质分子。
在连接构件为脂质连接臂时，其结构上可以包括多个端的结构，但其中至少有两端，所述亲脂末端(与脂质杂化物质连接)和所述亲水末端，并且亲脂末端为脂质分子。
脂质分子包括：脂肪酸类(fatty acids)，甘油脂类(glycerolipids)，甘油磷脂类(glycerophospholipids)，鞘脂类(sphingolipids)，固醇脂类(sterol lipids)，孕烯醇酮脂类(prenol lipids)，糖脂类(saccharolipids)，多聚乙烯类(polyketides),阳离子脂质(cationic lipids)和可电离脂质(ionizable lipids)。
可选地，所述脂质分子，可以为DSPE(二硬脂酰基磷脂酰乙醇胺)、二硬脂酰磷脂酰胆碱(DSPC)、1,2-二肉豆蔻酸甘油酯(DMG,1,2-Dimyristoyl-sn-glycerol)、1,2-双棕榈酸甘油酯(DPG,1,2-DIPALMITOYL-SN-GLYCEROL)、1,2-双棕榈酸甘油酯(DPyG,1,2-Diphytanoyl-sn-glycerol)、二酰基甘油(DAG)；甘油三酯(TAG),1,2-双棕榈酸甘油酯(DPG),1,1'-[(1R)-1-(羟基甲基)-1,2-乙二基]十八烷酸酯(DSG)，二花生酰基磷脂酰胆碱(DAPC),1-棕榈酰-2-月桂酰-sn-甘油-3-磷酸胆碱(DLPC),二肉豆蔻酰磷脂酰胆碱(DMPC),1,2-二油酰基卵磷脂(DOPC),二棕榈酸磷脂酰胆碱(DPPC),1,2-二棕榈酰-sn-甘油-3-乙基磷酸胆碱(DPePC)，磷脂酰胆碱(PC),磷脂酰乙醇胺(PE),磷脂酰甘油(PG),磷脂酰丝氨酸(PS),磷脂酰肌醇(PI)；神经酰胺(Cer)，鞘磷脂(SM)，胆固醇(Cho),胆固醇酯(CE),1,2-二豆蔻酰-SN-甘油-3-磷酸(DMPA),二月桂酰基磷脂酸(DLPA),磷酸胆碱(Phosphocholine)，溴化三甲基-2,3-二油酰氧基丙基铵(DOTAP),可电离脂质包括DLin-MC3-DMA、A6、OF-02、A18-ISO5-2DC18、98N12-5、9A1P9、C12-200、cKK-E12、7c1、G0-C14、L319、304O13、OF-Deg-Lin、306-OB12、306-Oi10和FTT5，及前述脂质分子衍生物和其药学上可接受的盐。所述PEG脂质可以为PEG及PEG衍生物与前述脂质的偶联物。
可选地，脂质分子可以为PEG化脂质，其中，PEG相对分子量为2000，PEG化脂质可以包括：PEG-DSPE(聚乙二醇-二硬脂酰基磷脂酰乙醇胺)、PEG-DMG(聚乙二醇-二肉豆蔻酸甘油酯)、前述脂质分子和PEG的偶联物及其药学上可接受的盐。所述PEG包括PEG和PEG末端为甲氧基或其他基团的PEG。
可选地，所述两亲性聚合物为链状或支化分子结构的聚合物，其至少存在一个具有亲水性的分子端，以及一个具有疏水性的分子端；
所述两亲性聚合物的结构为链状或支化分子结构，因为其链状或支化分子结构的特性，因此可以具有多个分支，多条链，但其具有至少两个端，一个为具有亲水性的分子端，一个为具有疏水性的分子端。
可选地，所述两亲性聚合物为直链分子结构，其一端为亲水性的分子端，另一端为具有疏水性的分子端。
上述，进一步限定两亲性聚合物为直链分子结构。
可选地，还包括纳米颗粒，其中，所述纳米颗粒被所述脂质杂化物质包覆于所述基于脂质的蛋白降解工具的核心处；
所述纳米颗粒包括亲水性颗粒、疏水性颗粒和无机纳米颗粒；
可选地，所述纳米颗粒的粒径为5-1000nm。
在另一种实施方试中，提供了一种特殊结构，即为纳米颗粒在核心处，脂质杂化物质包裹了纳米颗粒，构成复合结构。
因此，基于脂质的蛋白降解工具可以包括如下几种形态结构：
表5、基于脂质的蛋白降解工具的形态结构
此外，在另一种实施方式中，提供了一种特殊结构，即为纳米颗粒在核心处，脂质杂化物质包覆于纳米颗粒，构成复合结构，
其中，所述纳米颗粒被所述脂质杂化物质包覆于所述基于脂质的蛋白降解工具的核心处；
所述纳米颗粒(被包覆于脂质杂化物质中核心处的纳米颗粒)包括：
(1)亲水性颗粒，可以为，树枝状聚合物、超支化聚合物、以及前述亲水聚合物及其衍生物组成的各种纳米凝胶、以及纳米白蛋白；
(2)疏水性颗粒，可以为，聚乳酸-羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚己内酯(PCL)、聚碳酸酯(PMC)及其衍生物、乙交酯/丙交酯/己内酯/碳酸酯各类组合及组分的共聚物、聚氨酯(PU)、聚醚醚酮(PEEK)、聚甲基丙烯酸甲酯(PMMA)、聚乙烯醇(PVA)、聚乙烯(PE)、聚苯丙氨酸等各类疏水聚氨基酸及其衍生物制备的纳米颗粒；
(3)无机纳米颗粒，可以为，金纳米颗粒、碳纳米颗粒、硅纳米颗粒、氧化铁纳米颗粒、磷酸钙纳米颗粒、硫酸钡和碘化物造影剂、氮化铝纳米颗粒、氧化铝纳米颗粒、氧化钛纳米颗粒、铝铁合金颗粒、钛铁合金颗粒，均为具有稳定且低生物毒性的材料；(4)以及前述纳米颗粒组成的混合纳米颗粒。
其中，在纳米颗粒为亲水性颗粒或疏水性颗粒两种之一，并且其为聚合物时，由脂质杂化物质包覆后，也可以称为脂质多聚物(LPP,lipopolyplex)即在具有亲水或疏水性质的聚合物的纳米颗粒表面包覆脂质膜，以及阳离子纳米乳(Cationic nanoemulsion)即阳离子脂质构成的乳滴也在疫苗和药物递送中被广泛研究和应用。
可选地，在POI识别基团中，所述抗体为治疗性单克隆抗体、多特异性抗体、纳米抗体及前述抗体衍生物或抗体偶联药物；
所述多肽为具有POI特异性结合能力的多肽；
所述小分子为具有POI特异性结合能力的化合物；
可选地，所述小分子包括CDK4/6蛋白抑制剂帕博西尼(Palbociclib)的衍生物，BRD4蛋白抑制剂JQ1的衍生物,β-Amyloid蛋白探针AV-45的衍生物，匹兹堡化合物PiB的衍生物，Tau蛋白探针GTP1、PBB3的衍生物。
可选地，所述多肽包括ACE2、CD13、β-Amyloid的binding多肽；
可选地，所述抗体包括：CTX、NTZ、PTZ、CRLZ、INE、ATZ，和aducanumab，Miltuximab。
此外，本公开还提供了一种基于脂质的蛋白降解工具，在制备治疗和预防蛋白异常积累相关疾病的药物、疫苗及递送系统中的应用；其中，所述蛋白异常积累疾病包括肿瘤、免疫系统疾病、炎症和病原体感染、神经退行性疾病、血液系统疾病和代谢类疾病。
此外，本公开还提供了一种基于脂质的蛋白降解工具，在制备针对蛋白异常积累相关疾病及蛋白互作研究的检测产品和/或试剂盒中的应用；其中，所述蛋白异常积累疾病包括肿瘤、免疫系统疾病、炎症和病原体感染、神经退行性疾病、血液系统疾病和代谢类疾病。
此外，本公开还提供了一种基于脂质的蛋白降解工具的制备方法，其中，在所述基于脂质的蛋白降解工具为所述POI识别基团与所述脂质杂化物质偶联的蛋白降解工具时，其制备方法包括两种：
(1)将所述POI识别基团通过非共价键合到所述脂质杂化物质上；或者
(2)将所述POI识别基团基于偶联基团通过共价键合到所述脂质杂化物质上，从而构成所述基于脂质的蛋白降解工具。
可选地，所述基于脂质的蛋白降解工具还包括所述POI识别基团通过连接构件与所述脂质杂化物质连接所构成的蛋白降解工具；
其中，在所述基于脂质的蛋白降解工具为所述POI识别基团通过所述连接构件与所述脂质杂化物质连接所构成的蛋白降解工具时，其制备方法为：
(1)先将所述POI识别基团与所述连接构件偶联，构成偶联中间体；然后将所述偶联中间体连接到所述脂质杂化物质上，构成所述基于脂质的蛋白降解工具；或者
(2)先构建由所述连接构件作为外层且由所述脂质杂化物质作为核心的纳米复合结构；再将所述POI识别基团与所述纳米复合结构偶联，构成所述基于脂质的蛋白降解工具。
本公开通过将POI识别基团修饰于纳米颗粒的表面，或POI识别基团连接于连接臂，或POI识别基团修饰于连接臂并通过连接臂连接于纳米颗粒表面，POI识别基团组装后暴露于纳米颗粒或连接臂的外部，其中，纳米颗粒(NP,Nanoparticle)能够不依赖特殊设计的受体-配体匹配对进行细胞渗透，并且可以偶联小分子、多肽、核酸适配体、抗体等POI识别基团，特异性地进行蛋白质劫持和靶向降解，从而实现基于纳米颗粒的TPD工具(TPD-NP)的组装。这种便捷的基于纳米颗粒的TPD工具极易获得载药和组织特异性的靶向的能力，使得药物和蛋白降解联合治疗以及转化/精准医学成为可能。TPD-NP的发明和其机制的探索，大大扩展了TPD工具的范围，并为TPD和纳米递送领域提供了基础知识， 并且原则上可以在体内降解多种人类疾病相关的细胞外/膜相关/细胞内蛋白。
本公开首次系统地提出纳米颗粒介导的蛋白质降解，对于TPD和纳米递送提供了新的途径。本公开的纳米蛋白降解工具结构灵活，便于改造，可获得载药、靶向、穿越生物屏障的能力。本公开的纳米蛋白降解工具具有普适性，靶点可随意更换，三种组分均可以替换，应用场景广。本公开纳米蛋白降解工具所有组分可以是临床批准的材料，体内应用潜力高，有转化价值，本公开的纳米蛋白降解工具无须从头合成，为即用型平台，大大降低了开发和生产的复杂性和难度。相比PROTAC降解胞内蛋白，LYTAC降解胞外/膜蛋白，TPD-NP可以降解胞外/胞内/膜蛋白。另外，相对于LYTAC等胞外/膜蛋白降解工具，TPD-NP不需要额外设计辅助所劫持蛋白被内吞的结构。相比目前已有的TPD工具，本公开的纳米蛋白降解工具不需要特殊结构引导蛋白降解。纳米颗粒可以载药、可以设计成为可控释放，可以设计成为光热磁等协同治疗材料，可以成像、造影进而进行蛋白降解的协同治疗和诊疗一体化。NP的体液稳定性可以减少药物损失，提高成药潜力。
此外，本公开通过将POI识别基团修饰于脂质杂化物质的表面，并且POI识别基团组装后暴露于脂质杂化物质的外部，实现了基于脂质杂化物质的靶蛋白降解，使得构建蛋白降解工具的合成难度极大降低，通过“即插即用”模式，使得海量与靶蛋白具有结合力的化合物、多肽、抗体和核酸适配体等能够升级成为蛋白降解类药物，进而在传统脂质体(Liposome)、脂质纳米颗粒(LNP)相关领域，如mRNA疫苗、核酸递送载体、药物递送中发挥新的功能，从而实现在科研和工业应用中进行联合疗法开发。
而且，配体靶向的脂质类纳米颗粒(脂质杂化物质)作为蛋白降解工具的应用和其机制的探索尚属空白，该发明大大扩展了目前脂质类纳米颗粒的应用范围，并为TPD和纳米递送领域提供了基础知识，并且原则上可以在体内降解多种人类疾病相关的细胞外/膜相关/细胞内蛋白。
实施例
下面通过实施例进一步说明本公开，但是应当理解为，这些实施例仅仅是用于更详细地说明之用，而不应理解为用于以任何形式限制本公开。
表6、实施例1-13中所制备的蛋白降解工具的结构形态

实施例1
参考表6，本实施例中，对于第一降解工具进行了制备，包括POI识别基团与连接臂偶联，随后自组装。其中，POI识别基团为单抗药物尼妥珠(NTZ)，连接臂为NHS-PEG-DSPE(N-羟基琥珀酰亚胺修饰的聚乙二醇-二硬脂酰磷脂酰乙醇胺，或称DSPE-PEG-NHS)。
制备方法：
(1)预处理：将原始抗体的缓冲液置换为磷酸盐缓冲液PBS。本实施例中，采用已应用于临床治疗的EGFR单抗尼妥珠(NTZ,Nimotuzumab)。
取尼妥珠单抗溶液，用3kDa超滤管在4000×g离心2分钟，进而浓缩抗体，随后用PBS稀释；反复浓缩稀释3次后，缓冲液主要成分被置换为PBS，抗体溶液稀释后，通过NanoDrop one C仪器的IgG模式以A280处的吸光度测定蛋白质浓度，并通过BCA蛋白质定量试剂盒验证其结果。
(2)偶联反应：随后进行抗体和连接臂的偶联反应，抗体与NHS-PEG(2kDa)-DSPE通过抗体上的氨基与DSPE-PEG末端的NHS(N-羟基琥珀酰亚胺，N-Hydroxysuccinimide)基团反应。偶联反应在冰水混合物环境中起始，并加以氮气保护。为了提高分散性，可以选择将DSPE-PEG在PBS缓冲液中以40kHz超声处理3分钟，随后将相应摩尔浓度和体积(摩尔比例不限于1:1，参考抗体偶联药物)的NHS-PEG-DSPE PBS溶液添加到以800rpm(每分钟转数)搅拌的抗体PBS溶液中；然后搅拌反应在4℃，20rpm旋转器上孵育24小时(为了保持抗体活性更加倾向低转速和4度进行)。
其中，偶联方法包括但不限于非定点偶联和定点偶联。非定点偶联包括氨基偶联、羧基偶联、桥接巯基偶联。定点偶联包括点击化学反应、硒键偶联、丝氨酸偶联、半胱氨酸偶联、非天然氨基酸偶联、酶催化后偶联、糖位点偶联。
(3)纯化：然后将反应混合物用50kDa离心超滤管超滤浓缩3次，随后使用PBS定容至100μL。蛋白质浓度由NanoDrop one C仪器通过IgG模式以A280处的吸光度确定，并通过BCA蛋白质定量试剂盒验证其结果。
实验结果：
本公开的图4示出了本实施例的合成过程。其中，抗体与NHS-PEG-DSPE通过抗体上的氨基与DSPE-PEG末端的NHS基团进行偶联反应，得到缀合物NTZ-PEG-DSPE
本公开的图5示出了本实施例的考马斯亮蓝染色结果。考马斯亮蓝染色结果显示NTZ抗体在与连接臂反应得到纳米颗粒NTZ-PEG-DSPE后，质量增大，迁移速率减慢，考马斯亮蓝与蛋白结合后显色条带靠上，表明POI识别基团成功连接到了PEG-DSPE臂上。
本公开的图6示出了本实施例的蛋白降解效果的Western blot蛋白电泳图。结果显示NTZ-PEG-DSPE纳米颗粒可以有效降解靶蛋白EGFR。
需要说明的是，所有实施例中细胞培养基体积均相同，其所接受的处理的摩尔浓度单位为μM(μmol/L),nM(nmol/L)。所有对照组所用的摩尔浓度与降解工具组保持一致，例如NTZ组和NTZ-NP组其抗体NTZ的摩尔浓度和体积均相同，同时，不含抗体的NP组和NTZ-NP组的脂质杂化物质摩尔浓度和体积均相同。其中NP组指所在实施例中未连接POI识别基团的纳米颗粒。所有涉及多浓度测试的实施例中，其对照POI识别基团组或对照脂质杂化物质组均以浓度测试组中最高浓度处理的相等摩尔浓度处理。所有蛋白电泳图以GAPDH或VIN(vinculin)为内参。
实施例2
参考表6，本实施例中，对于第二降解工具进行了制备，包括POI识别基团与连接臂连接，随后自组装形成第二降解工具。在该第二降解工具的终产品中，POI识别基团被暴露在整体外部。其中，在实施例2a中，POI识别基团为BRD4蛋白的抑制剂JQ1的氨基衍生物JQ1-NH2，其氨基用于纳米颗粒偶联，BRD4为肿瘤表观遗传和增殖调控分子。在实施例2b中，POI识别基团为EGFR单抗尼妥珠NTZ,其氨基用于纳米颗粒的偶联。在实施例2a和实施例2b中，纳米颗粒为NHS-PEG-PLGA(N-羟基琥珀酰亚胺-聚乙二醇-聚乳酸-羟基乙酸共聚物)有机相转水相形成的纳米颗粒，NHS暴露于纳米颗粒外部。
其中，偶联方法包括但不限于非定点偶联和定点偶联，非定点偶联如氨基偶联、羧基偶联、桥接巯基偶联。定点偶联包括点击化学反应、硒键偶联、丝氨酸偶联、半胱氨酸偶联、非天然氨基酸偶联、酶催化后偶联、糖位点偶联。
实验方法：
本实施例2a中，POI识别基团选择了BRD4蛋白的抑制剂JQ1的氨基衍生物JQ1-NH2，通过JQ1-NH2连接到PEG-PLGA纳米颗粒的PEG末端NHS基团上。
所用PEG-PLGA纳米颗粒通过聚合物NHS-PEG(3kDa)-PLGA(5kDa)有机相转水相，自组装成纳米颗粒。浓度测定采用化合物紫外吸收峰的吸收标准曲线矫正。
使用终浓度为500nM，1μM,1.5μM的JQ1、与JQ1等价摩尔量的JQ1-NP和与JQ1等价摩尔量的NP处理M231人乳腺癌肿瘤细胞24小时，随后收集细胞总蛋白，检测蛋白表达。
本实施例2b中，POI识别基团选择了NTZ单抗，通过NTZ单抗赖氨酸残基的氨基与PEG-PLGA纳米颗粒的PEG末端NHS基团偶联，以获得NTZ-NP2。
所用PEG-PLGA纳米颗粒通过聚合物NHS-PEG(3kDa)-PLGA(5kDa)有机相转水相，自组装成纳米颗粒。浓度采用Nanodrop one C仪器的IgG模式以A280处的吸光度测定和BCA蛋白质定量试剂盒验证其结果。
实验结果：
本公开的图7为第二降解工具的合成过程示意图。
本公开的图8中的结构式为JQ1-NH2的结构，其末端游离NH2用于与PEG-PLGA纳米颗粒偶联。图8中动态光散射DLS结果显示纳米颗粒粒径约为100nm。平均分散系数PDI约为0.2，表明纳米颗粒较为均一。
本公开的图9示出了实施例2中第二降解工具JQ1-NP蛋白电泳结果。蛋白电泳结果显示JQ1偶联纳米颗粒后，细胞BRD4蛋白水平下调，而其溶剂PBS对照组、单纯JQ1小分子组和单纯纳米颗粒对照组均无BRD4降解效果。
本公开的图10示出了实施例2中第二降解工具NTZ-NP2蛋白电泳结果。蛋白电泳结果显示500nM NTZ抗体偶联纳米颗粒NTZ-NP2处理24小时后，M231细胞EGFR蛋白水平下调，而其溶剂PBS对照组、单纯EGFR单抗组和单纯纳米颗粒对照组均无EGFR降解效果。
实施例3
参考表6，本实施例中，对于第三降解工具进行了制备，包括POI识别基团与连接臂偶联后，再与纳米颗粒进行偶联，形成复合结构。其中，POI识别基团为NTZ抗体。通过抗体上的氨基与NHS-PEG-DSPE连接臂偶联，获得NTZ-PEG-DSPE偶联中间体，随后NTZ-PEG-DSPE偶联中间体沉淀于PLGA纳米颗粒表面产生复合结构，从而最终构成了内层为纳米颗粒核心(PLGA)、中间层为两亲嵌段聚合物(PEG-DSPE)、外围为NTZ抗体的蛋白降解工具，即“NTZ-NP”。
1、制备的总体方法：POI识别基团和连接臂PEG-DSPE的偶联采用第一降解工具中的偶联方法。POI识别基团-PEG-DSPE与PLGA纳米颗粒偶联，采用PLGA有机相转水相，POI识别基团-PEG-DSPE在水相中与PLGA自组装沉淀于PLGA核心表面。
制备方法：
(1)配制NTZ-PEG(2kDa)-DSPE的PBS溶液(摩尔比PLGA：NTZ-PEG-DSPE＝1：1)
(2)将溶解在DMF中的PLGA(15kDa)逐滴滴入(每滴2μL，每滴间隔5秒)到600rpm搅拌的DSPE-PEG-NTZ的PBS溶液中，随后在室温下持续搅拌30分钟，完成PLGA与DSPE-PEG-NTZ的自组装。
(3)将溶液通过截留分子量3kDa的超滤管，离心浓缩5次，每次4分钟，去除未结合的成分并用PBS补足所需体积。
(4)通过NanoDrop one C(Thermofisher)以IgG模式测定蛋白质浓度，并通过BCA蛋白质测定试剂盒进行蛋白浓度验证。
2、实验1：物质结构验证
本公开的图11示出了第三降解工具的合成过程。其中，通过抗体上的氨基与连接臂偶联，获得偶联中间体，随后偶联中间体沉淀于纳米颗粒表面产生复合结构。
本公开的图12示出了本实施例中所制备的第三降解工具的形态结构，其为内层为纳米颗粒核心(PLGA)、中间层为两亲嵌段聚合物(PEG-DSPE)、外围为NTZ抗体的蛋白降解工具。
本公开的图13示出了本实施例中所制备的第三降解工具的动态光散射结果和透射电镜照片。如图13所示，纳米颗粒合成后通过动态光散射测得该纳米颗粒粒径约为150nm，平均分散系数(PDI)为0.153，说明所得的纳米药物具有较好的均一性。通过透射电子显微镜测得纳米颗形貌如图所示为球形，比例尺为100nm。上述结果证明成功获得了第三降解工具。
3、实验2：蛋白降解功能的验证
(1)实验2方法：尼妥珠NTZ为人EGFR单抗，EGFR为肿瘤增殖信号受体和表面标志物。NTZ-NP以500nM的终浓度处理MDA-MB-231(M231)人乳腺癌细胞，HeLa人宫颈癌细胞，U87人脑胶质瘤细胞24小时，随后检测EGFR蛋白表达。
(2)实验2结果：
本公开的图14示出了实施例3中第三降解工具(NTZ-NP)在人乳腺癌M231细胞、人宫颈癌HeLa细胞、人脑胶质瘤U87细胞蛋白降解效果的蛋白电泳结果。如图14所示，500nM NTZ或NTZ-NP偶联物处理M231细胞、HeLa细胞和U87细胞24小时后，蛋白电泳显示EGFR表达明显降低。上述结果证明该纳米粒子能够在乳腺癌M231细胞、宫颈癌HeLa细胞、胶质瘤U87细胞多种肿瘤细胞中降解膜受体蛋白EGFR。
本公开的图15示出了M231细胞经第三降解工具处理后的免疫荧光染色结果。如图15所示，500nM抗体剂量的NTZ-NP处理M231细胞24小时后，通过免疫荧光染色，共聚焦显微镜检测细胞表面EGFR的表达情况,结果显示EGFR表达明显降低(比例尺＝10μM，箭头标注为EGFR信号，DAPI为细胞核染料，Merge为EGFR和DAPI叠加信号)。
本公开的图16示出了M231细胞经第三降解工具处理后的固定视野活细胞时间序列拍摄绿色荧光标记的EGFR蛋白降解结果。如图16所示，为了进一步明确EGFR蛋白降解，我们采用了EGFP绿色荧光蛋白融合到EGFR的胞内结构域(终止密码子前)转染的M231细胞进行实验。固定视野下，在活细胞培养环境中，共聚焦显微镜连续拍摄，结果表明EGFP标记融合的EGFR的绿色荧光信号随着时间的增加逐渐降低(比例尺＝25μm)。
本公开的图17示出了HepG2细胞经第三降解工具处理后的CCK8检测细胞活力结果。如图17显示，500nM抗体剂量的NTZ-NP处理人肝癌细胞HepG2细胞24h后，经CCK8细胞活力测定试剂盒检测，其细胞增殖受到显著抑制， ***p<0.001具有统计学显著差异。
4、实验3：检测处理时间和浓度与蛋白降解效果之间的关联
(1)实验3方法：实施例中采用了不同浓度NTZ-NP处理M231细胞24小时后，检测其EGFR蛋白降解情况，以探索有效降解浓度，同时采用500nM NTZ-NP处理M231细胞不同时间。
(2)实验3结果：本公开的图18示出了实施例3中第三降解工具的EGFR蛋白表达，其中a图为不同浓度的NTZ-NP处理24小时后的EGFR蛋白表达，结果表明EGFR被有效降解的浓度可以低至10nM，理论上具有较好的成药性。b图为500nM NTZ-NP处理M231细胞不同时间后收取总蛋白进行蛋白电泳，其中从24小时起，培养基被更换为无处理的新鲜培养基，以便观察靶蛋白蛋白动态恢复情况。可见单次处理最佳降解时间为24-48小时，72小时后逐渐恢复。逐渐恢复的表现，符合蛋白降解工具“临时性降解”的理论基础。
5、实验4：偶联至PLGA核心上的NTZ-PEG(2kDa)-DSPE偶联中间体与无NTZ连接的空白连接臂的比例与EGFR降解效果的关系。
(1)实验4方法：通过在NTZ-PEG-DSPE与PLGA自组装时混入不同比例的不含抗体的PEG-DSPE连接臂，调整NTZ-PEG-DSPE所占的比例，随后500nM(等价NTZ抗体的摩尔浓度)的不同NP处理M231细胞24h。
(2)实验4结果：本公开的图19示出了实施例3中第三降解工具的EGFR蛋白表达，其研究了包含POI识别基团和连接臂构成的偶联中间体与不含POI识别基团的空白连接臂之间不同的比例对降解效果的影响。结果显示相同抗体含量情况下，NTZ-PEG-DSPE中间体与空白连接臂比例为1:1时，即NTZ-PEG-DSPE偶联中间体含量为50％左右时，蛋白降解效果较好。上述结果表明，与50％的NTZ-PEG-DSPE臂的实验组相比，100％的NTZ-PEG-DSPE臂的实验组可能由于POI识别基团NTZ产生位阻，进而影响蛋白降解。
实施例4
参考表6，本实施例中，对于第三降解工具进行了制备，包括POI识别基团与连接臂偶联后，再与纳米颗粒进行偶联，形成复合结构。其中，POI识别基团为：NTZ抗体，连接臂为：聚乙二醇PEG，纳米颗粒为复合结构，纳米颗粒为无机材料金纳米颗粒(AuNP)，由此构成复合结构，即“NTZ-AuNP”。
实验方法：制备第三降解工具
POI识别基团和连接臂NHS-PEG-SH(N-羟基琥珀酰亚胺-聚乙二醇-巯基)的偶联采用第一降解工具中的偶联方法。POI识别基团-PEG-DSPE与金纳米颗粒偶联，采用巯基和金纳米颗粒之间自发形成金-硫键的方式偶联。偶联方法包括通过活性基团发生共价反应或通过非共价偶联。
具体方法：取尼妥珠单抗溶液，用3kDa超滤管在4000×g浓缩并其置换为0.1M pH＝8.0碳酸氢钠(NaHCO3)溶液。在0.1M,pH＝8.0的NaHCO3溶液中制备1mg/mL NHS-PEG(2kDa)-SH溶液，并立即以1:1的摩尔比加入尼妥珠单抗，在冰上600rpm搅拌，逐滴将PEG加入，并在4℃搅拌下孵育过夜，获得NTZ-PEG-SH连接臂。
AuNP与NTZ-PEG-SH(摩尔量比为1：1)通过-SH和金发生金硫键反应偶联。具体而言，在PBS中室温600rpm搅拌2小时,之后4℃反应过夜。然后进行超滤浓缩。通过NanoDrop one C(Thermofisher)以IgG模式以A280处的吸光度测定蛋白质浓度，并通过BCA蛋白质测定试剂盒进行验证。
实验结果：
本公开的图20示出了实施例4中第三降解工具(NTZ-AuNP)的结构示意图。
本公开的图21示出了实施例4中第三降解工具(NTZ-AuNP)的蛋白降解效果电泳结果。如图21所示，M231细胞经NTZ-AuNP及对照处理24h后，EGFR蛋白表达明显降低。
实施例5
参考表6，本实施例中，对于第三降解工具进行了制备，包括POI识别基团与连接臂偶联后，再与纳米颗粒进行偶联，形成复合结构。其中，POI识别基团为：具有CD13蛋白或ACE2蛋白或β-amyloid 1-42寡聚体结合能力的多肽，连接臂为：两亲聚合物(PEG-DSPE)，纳米颗粒为PLGA，由此构成复合结构，即“CD13-NP”或“ACE2-NP”或“AB-NP”。
CD13为引起普通感冒的冠状病毒受体和肿瘤标志物，其结合多肽AP-1来源于文献，序列为NH2-YVEYHLC-COOH。ACE2为冠状病毒受体，COVID19新冠肺炎的病毒SARS-Cov2通过ACE2蛋白受体侵染细胞，其结合多肽序列NH2-CSPLRYYPWWACT-COOH来源于文献。β-amyloid 1-42寡聚体(β-amyloid oligomer)为阿尔兹海默症的潜在致病蛋白，其为胞外蛋白，前体表达于细胞膜上，剪切后分泌至胞外形成寡聚体和纤维，其中寡聚体被认为毒性最大，作为胞外蛋白β-amyloid oligomer的积累带来神经炎症等病理风险，其结合多肽序列NH2-KLVFF-COOH来源于文献。
实验方法：制备第三降解工具
将多肽上的游离-NH2/-COOH/-SH基团，或修饰的叠氮基、炔基、马来酰亚胺或NHS等活性残基，用于对应的DSPE-PEG的活性基团的偶联。多肽的偶联方法为通过一般抗体偶联药物的偶联方法，包括但不限于位点非特异性偶联和特异性偶联，以及最常见的氨基偶联、点击化学反应、羧基偶联、巯基偶联、硒键偶联。
(1)多肽和1当量(eq，摩尔比)的DSPE-PEG-活性基团粉末在氮气保护下溶解在2mL DMF溶剂中(溶剂无特殊要求，可替换为其他任何有助于多肽溶解的溶剂)，随后体系中添加1.5的三乙胺。
(2)在37℃水浴中以800rpm搅拌12小时以上，将反应溶液通过2kDa(截留分子量无特殊要求，能够区分偶联成功与否即可)透析12小时以上，去除未结合的物质，然后将产物冻干，获得多肽-PEG-DSPE。
(3)配制多肽-PEG-DSPE的PBS溶液(质量比：PLGA:多肽-PEG-DSPE＝1：4)。
(4)将溶解在DMF中的PLGA(15kDa)逐滴滴入(每滴2μL，每滴间隔5秒)到600rpm搅拌的多肽-PEG-DSPE PBS溶液中，并反应在室温下继续搅拌30分钟。
(5)将溶液通过3kDa超滤管离心浓缩5次，每次用PBS稀释后离心4分钟以去除未结合的物质。获得多肽-NP，之后通过BCA蛋白质测定试剂盒测定浓度。
实验结果：
(1)将所得的产物溶在氘代DMSO中，进行核磁共振波谱仪检测。
本公开的图22示出了本实施例中第三降解工具(ACE2-NP)中POI识别基团与连接臂连接产物(ACE2-PEG-DSPE)的核磁共振氢谱(1H NMR)。本公开的图23示出了本实施例中第三降解工具(CD13-NP)中POI识别基团与连接臂连接产物(CD13-PEG-DSPE)的核磁共振氢谱(1H NMR)。
如图22和图23所示，核磁共振氢谱(1H NMR)结果表明本实施例的肽段与PEG-DSPE成功连接。
(2)本公开的图24示出了本实施例中第三降解工具(ACE2-NP)处理被GFP标记ACE2的293细胞后的流式细胞术检测GFP结果图。如图24所示，293T细胞转染ACE2-GFP载体(GFP融合到细胞内结构域,终止密码子前)后，经10μM ACE2结合肽(ACE2)或其NP偶联物(ACE2-NP)处理24h后，流式细胞仪结果显示ACE2-GFP阳性群体降低。
本公开的图25示出了本实施例中第三降解工具(CD13-NP)处理人肝癌细胞HepG2后的蛋白电泳结果图。如图25所示，2μM CD13结合肽(CD13)或其NP偶联物(CD13-NP)处理24h后，HepG2细胞中CD13的表达有明显降低。
本公开的图26示出了本实施例中第三降解工具(AB-NP)处理人胶质细胞后，共聚焦显微镜检测FITC绿色染料标记的β-amyloid 1-42寡聚体的细胞内吞的实验结果。1μM的FITC荧光素标记的5μM的β-amyloid oligomer寡聚体和AB-NP在人胶质细胞中共培养4小时，随后用PBS洗去胞外溶液。如图26所示，被FITC标记的β-amyloid oligomer寡聚体经AB-NP处理后，更多的β-amyloid被吞噬，并且与溶酶体染料LysoTracker标记的溶酶体具有共定位(比例尺＝100μm)，溶酶体理论上可以降解β-amyloid以消除因β-amyloid胞外积累带来的毒性。
实施例6
参考表6，本实施例中，对于第三降解工具进行制备，包括POI识别基团与连接臂偶联后，再与纳米颗粒进行偶联，形成复合结构。其中，POI识别基团为小分子药物Palbociclib或小分子探针AV-45，连接臂为两亲聚合物(PEG-DSPE)，纳米颗粒为PLGA。
实验1：
POI识别基团为CDK4抑制剂Palbociclib,其纳米颗粒偶联物为Palb-NP。CDK4为细胞周期蛋白依赖性激酶，广泛参与到细胞衰老肿瘤发生中。
制备方法：
(1)将Palbociclib与NHS-PEG-DSPE，在室温氮气保护下，搅拌反应24小时，得到Palb-PEG-DSPE连接臂反应混合物，随后透析冻干。
(2)Palb-PEG-DSPE-(PLGA)即Palb-NP的合成方式为PLGA有机相转水相自组装，与实施例3的方法相同，PLGA溶解于DMF中，Palb-PEG-DSPE溶解于PBS中。
实验2：
POI识别基团为β-amyloid探针AV-45,其纳米颗粒偶联物为AV45-NP。β-amyloid为阿尔兹海默症的潜在致病蛋白，其为胞外蛋白，前体表达于细胞膜上，剪切后分泌至胞外形成寡聚体(β-amyloid 1-42oligomer)和纤维，其中寡聚体被认为毒性最大，作为胞外蛋白β-amyloid oligomer的积累带来神经炎症等病理风险，同位素标记的AV-45是一种临床商用β-amyloid探针。本实施例选用的为不含同位素的AV-45衍生物AV-45-SH，AV-45-SH具有蓝色自发荧光，便于细胞追踪，其巯基SH用于和Mal-PEG-DSPE偶联。
制备方法：将带有巯基的AV45-SH与Mal-PEG2K-DSPE结合，再与PLGA结合。
(1)AV45-PEG-DSPE制备：将AV45-SH与Mal-PEG2K-DSPE溶于DMSO，摩尔比1.2:1，避光37℃水浴24h。
(2)AV45-NP制备：避光透析24h，以去除游离的AV45-SH，随后连接PLGA核心，连接方法采用本实施例实验1的方法，PLGA有机相转水相自组装，AV45-PEG-DSPE在水相与PLGA自组装。通过AV45的紫外吸收进行定量。
(3)β-amyloid劫持实验：10μM的FITC绿色染料标记的β-amyloid 1-42多肽制备的寡聚体与10μM的AV45-NP共孵育，随后通过PBS洗掉未进入细胞的的处理物，进行共聚焦显微镜成像，检测β-amyloid 1-42寡聚体的绿色荧光，绿色荧光强则表明被劫持进入细胞量更多，理论上更多的吞噬和溶酶体转移能带来更好的降解，以减轻胞外β-amyloid oligomer的积累带来的毒性，溶酶体染料lysoTracker用来标记溶酶体。
实验结果：
浓度测定采用核磁计算接枝率，并用紫外吸收标准曲线矫正。
本公开的图27示出了实施例6中第三降解工具(Palb-NP)中POI识别基团与连接臂连接产物(Palb-PEG-DSPE)的核磁共振氢谱(1H NMR)。图27中的核磁共振氢谱表明小分子药物Palbociclib成功偶联至PEG-DSPE。
本公开的图28示出了实施例6中第三降解工具(Palb-NP)的蛋白降解效果的蛋白电泳图。2.5μM Palbociclib或Palbociclib偶联的纳米颗粒(Palb-NP)处理M231细胞24小时，收蛋白检测CDK4蛋白表达，图28中的结果表明Palb-NP能有效抑制肿瘤增殖。
本公开的图29示出了实施例6中第三降解工具处理M231细胞后结晶紫染色克隆形成的实验结果。3.5μM Palbociclib或Palb-NP处理M231细胞48小时，随后继续培养7日，进行结晶紫染色克隆形成实验。图29中的结果表明Palb-NP抑制肿瘤增殖效果优于Palbociclib。
本公开的图30为AV45-NP结构示意图。
本公开的图31示出了实施例6中第三降解工具(AV45-NP)中POI识别基团与连接臂连接产物(AV45-PEG-DSPE)的核磁共振氢谱(1H NMR)，图31中的核磁共振氢谱表明小分子药物β-amyloid探针AV-45成功偶联至PEG-DSPE。
本公开的图32示出了实施例6中第三降解工具(AV45-NP)处理人胶质细胞后，共聚焦显微镜检测FITC绿色染料标记的β-amyloid 1-42寡聚体的细胞内吞的实验结果。10μM AV-45或AV45-NP与10μM FITC绿色荧光染料标记的β-amyloid寡聚体在培养基中共孵育处理人胶质细胞12小时。图32的共聚焦显微镜结果显示AV45-NP组FITC绿色荧光信号更强，AV45自发荧光更强，并与溶酶体共定位，表明AV45-NP劫持更多β-amyloid进入人胶质细胞，可能促进其胞内降解。而与AV45-NP组相比，AV45单纯处理细胞后，其自发荧光极为微弱，也表明AV45-NP具有更好的细胞内化效率(图中比例尺为10μm)。
实施例7
本实施例中，对于第三降解工具进行了制备，分别得到CTX-NP、PTZ-NP、ATZ-NP、CRLZ-NP和NTZ/INE-NP，包括POI识别基团与连接臂偶联后，再与纳米颗粒进行偶联，形成复合结构。其中，POI识别基团、连接臂和纳米颗粒为PLGA分别如表6中所示。
制备方法：根据实施例1中所提供的制备方法合成CTX-PEG-DSPE、PTZ-PEG-DSPE、ATZ-PEG-DSPE、CRLZ-PEG-DSPE和INE-PEG-DSPE，并利用实施例3中的制备方法合成CTX-NP、PTZ-NP、ATZ-NP、CRLZ-NP和INE-NP，NTZ/INE-NP由等摩尔量的NTZ-PEG-DSPE与INE-PEG-DSPE共同自组装于PLGA而获得，其结构参考图8。
实验结果：
1、本公开的图33示出了实施例7中第三降解工具(CTX-NP)的蛋白降解效果的蛋白电泳图。如图33所示，500nM CTX-NP处理M231细胞24h后，蛋白电泳结果显示受体蛋白EGFR的表达量明显降低。
2、本公开的图34示出了实施例7中第三降解工具(PTZ-NP)的蛋白降解效果的共聚焦显微镜图。如图34所示，500nM PTZ-NP处理HepG2细胞24h后，HER2免疫荧光染色，通过共聚焦显微镜发现PTZ-NP组HER2受体蛋白的表达明显降低(比例尺＝10μm,箭头标注的膜结构为HER2)。
3、本公开的图35示出了实施例7中第三降解工具(ATZ-NP)的蛋白降解效果的蛋白电泳图。如图35所示，500nM ATZ-NP处理过表达PD-L1的293T细胞24h后，蛋白电泳结果显示PD-L1受体蛋白的表达明显降低。
4、本公开的图36示出了实施例7中第三降解工具(CRLZ-NP)的蛋白降解效果的蛋白电泳图。如图36所示，500nM CRLZ-NP处理过表达PD-1的JURKAT细胞24h后，蛋白电泳结果显示PD-1受体蛋白的表达明显降低。
5、本公开的图37示出了实施例7中第三降解工具(INE/NTZ-NP)的蛋白降解效果的蛋白电泳图。如图37所示，500nM INE-NP或NTZ/INE-NP处理HER2表达的293T细胞24h后，蛋白电泳结果显示HER2和EGFR受体蛋白的表达明显降低。这表明本公开能够通过简单的“即插即用”的方式获得多特异性靶向的能力，具有较好的应用潜力。
实施例8
1、实验1：NTZ-NP对MDA-MB-231乳腺癌皮下肿瘤移植模型的动物体内治疗效果。
本实施例采用了实施例3中获得的可选的第三降解工具，其中POI识别基团为NTZ，连接臂为DSPE-PEG,纳米颗粒核心为PLGA。本实施例中探究了NTZ-NP对MDA-MB-231(M231)乳腺癌皮下肿瘤移植模型的动物体内治疗效果。在周龄6-8周雌性裸鼠接种M231肿瘤的第10日开始隔日给药(15mg/kg)，连续监测小鼠肿瘤的体积(肿瘤长a宽b,体积计算公式：1/2ab2)，以及体重变化。给药两周，共计7次后，最后一次给药次日安乐死小鼠，分离肿瘤组织，肿瘤组织进行蛋白电泳以及免疫荧光检测。
实验结果：
本公开的图38a示出了NTZ-NP处理M231细胞裸鼠皮下瘤模型期间裸鼠肿瘤的体积。如图38a实验结果显示，NTZ-NP组相比溶剂对照PBS组，等摩尔量NTZ抗体组和等摩尔量无抗体的纳米颗粒组，肿瘤体积增殖受到明显抑制。
图38b为分离肿瘤组织后蛋白电泳检测EGFR蛋白表达水平，可见EGFR显著下调。每种处理完整的肿瘤组织取自不同的5只实验鼠并进行蛋白电泳和相对其自身内参的灰度统计，统计结果*p<0.05具有统计学差异。
图38c为NTZ-NP处理期间小鼠体重的变化，结果显示体重均未见明显变化。
图39为NTZ-NP处理结束后分离肿瘤组织进行免疫荧光染色检测EGFR的结果。
图40为NTZ-NP处理结束后分离肿瘤组织进行免疫荧光染色检测凋亡标志物的结果。其中，分离肿瘤组织后石蜡包埋切片，随后免疫荧光染色，检测EGFR和凋亡核心信号cleaved caspease-3免疫荧光染色结果，可见NTZ-NP组EGFR信号降低，凋亡信号上升。结果表明该NTZ-NP组纳米颗粒能够降解小鼠肿瘤细胞表面的生长因子受体蛋白EGFR，NTZ-NP对小鼠皮下肿瘤具有治疗效果，显示出体内应用潜力。
2、实验2：NTZ-NP纳米蛋白降解工具对小鼠的急性肝肾毒性研究。
本实施例采用了实施例3中获得的可选的第三降解工具，其中POI识别基团为NTZ，连接臂为DSPE-PEG,纳米颗粒核心为PLGA。本实施例中探究了NTZ-NP纳米蛋白降解工具对小鼠的急性肝肾毒性。将该纳米粒子和溶剂对照PBS，等摩尔量的NTZ抗体，等摩尔量的不含NTZ抗体的NP(10mg/kg)，通过尾静脉注射给白鼠，在注射后12小时取血，通过试剂盒进行血液生化指标检测。
实验结果：
本公开的图41示出了实施例8中第三降解工具(NTZ-NP)单次给药后白鼠的血液肝功能肾功能指标检测情况。如图41所示，实验结果显示血浆尿素(BUN)、尿酸(UA)、肌酐(CR)、白蛋白(ALB)、碱性磷酸酶(ALP)、丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)等相关指标未见明显异常，表明该纳米粒子对小鼠无明显的肝肾代谢毒副作用。
3、实验3：NTZ-NP是否可以通过简单的“即插即用”获得血脑屏障穿越能力和肿瘤靶向能力。
本实施例采用了实施例3中获得的可选的第三降解工具，其中POI识别基团为NTZ，连接臂为DSPE-PEG,纳米颗粒核心为PLGA。
本实施例中探究了NTZ-NP是否可以通过简单的“即插即用”获得血脑屏障穿越能力和肿瘤靶向能力。
将无法通过血脑屏障，也无法靶向肿瘤细胞的近红外染料DIR包裹于PLGA核中(DIR和PLGA在有机相互溶后，滴加进入NTZ-PEG-DSPE水相溶液中自组装，随后纯化浓缩)获得载有DIR的NTZ-NP。
在自组装NTZ-PEG-DSPE臂到PLGA核的时候，加入20％具有血脑屏障穿越能力的Angiopep2-PEG-DSPE,一同与载有DIR的PLGA自组装，随后纯化浓缩，获得载有DIR的Ang-NTZ-NP(其中angiopep-2多肽是一种代表性的血脑屏障穿越性多肽和肿瘤靶向多肽，其多肽序列(NH2-TFFYGGSRGKRNNFKTEEY-COOH)来源于文献。
静脉注射等摩尔量的两种纳米颗粒(抗体量10mg/kg，DIR量0.25mg/kg)给荷瘤(人脑胶质瘤原位移植)裸鼠，通过小动物成像仪IVIS系统，对比DIR荧光情况，用来显示血脑屏障穿越能力和肿瘤靶向聚集效果。
实验结果：
图42示出了实施例8中第三降解工具(NTZ-NP)通过简单的自组装获得血脑屏障穿越能力和肿瘤靶向能力，并装载了荧光染料DIR进行示踪，通过小动物成像仪检测DIR荧光的脑部聚集情况和统计图。
图42的左图显示当胶质瘤荷瘤鼠被注射含有20％Ang-2臂的纳米降解工具Ang-NTZ-NP时，脑部DIR染料信号聚集明显，右图为3次生物学重复实验统计，右图显示Ang-NTZ-NP脑内聚集水平明显提高，具有显著性差异。
结果证明，该纳米粒子具有载药能力，也可以被简单地通过即插即用进行修饰自组装，用以获得血脑屏障穿越能力和肿瘤靶向能力。
4、实验4：研究NTZ-NP是否可以通过简单的“即插即用”获得血脑屏障穿越能力和肿瘤靶向能力，并对原位肿瘤模型进行靶蛋白EGFR的蛋白降解和肿瘤抑制杀伤，并研究体内治疗潜力。
本实施例采用了实施例3中获得的可选的第三降解工具，其中POI识别基团为NTZ，连接臂为DSPE-PEG,纳米颗粒核心为PLGA。
本实施例中探究了NTZ-NP是否可以通过简单的“即插即用”获得血脑屏障穿越能力和肿瘤靶向能力，并对原位肿瘤模型进行靶蛋白EGFR的蛋白降解和肿瘤抑制杀伤，研究体内治疗潜力。
制备方法如本实施例实验3所述，在自组装NTZ-PEG-DSPE臂到PLGA核的时候，加入20％具有血脑屏障穿越能力的Angiopep2-PEG-DSPE,一同与PLGA自组装，随后纯化浓缩，获得的Ang/NTZ-NP。
在人胶质瘤移植给6-8周龄雌性裸鼠后15日，进行10mg/kg静脉给药，等摩尔量的对照和等体积溶剂对照也被同时尾静脉注射。每两日一次共计给药5次，完成给药周期后，次日安乐死小鼠，取脑组织石蜡包埋，切片进行免疫组化染色，研究肿瘤增殖标志物PCNA和NTZ的靶蛋白EGFR的蛋白降解效果。
本公开的图43示出了实施例8第三降解工具(NTZ-NP)通过图42的组装方式给与胶质瘤原位动物模型后脑肿瘤解剖后的免疫组化检测EGFR和增殖标志物PCNA结果图。结果显示，溶剂对照PBS组和NTZ-NP组EGFR和PCNA强于ANG/NTZ-NP组。表明该纳米蛋白降解工具具有较好的应用潜力(箭头标注为代表性阳性信号。比例尺为50μm)。
实施例9
参考表6，本实施例中，对于实施例9中的降解工具进行了制备，包括POI识别基团与脂质杂化物质偶联，其中POI识别基团为单抗药物尼妥珠(NTZ)；脂质杂化物质为PEG(聚乙二醇)化脂质体，即脂质体(liposome)含有PEG暴露于脂质杂化物质表面，其PEG末端具有活性反应位点NHS(N-羟基琥珀酰亚胺)用于和POI识别基团NTZ抗体的氨基偶联。本实施例获得的产物结构为NTZ-PEGlipo。
制备方法：
(1)预处理：将原始抗体的缓冲液置换为PBS。本实施例中，采用已应用于临床治疗的EGFR单抗尼妥珠(NTZ,Nimotuzumab)。
取尼妥珠单抗溶液，用3kDa超滤管在4000×g离心2分钟，进而浓缩抗体，随后用PBS稀释；反复浓缩稀释后，缓冲液被置换为PBS，稀释后通过NanoDrop one C仪器的IgG模式以A280处的吸光度测定蛋白质浓度，并通过BCA蛋白质定量试剂盒验证其结果。
(2)脂质杂化物质制备：使用的脂质杂化物质为脂质体，由氢化大豆卵磷脂(HSPC)、胆固醇(CHO，cholesterol)、DSPE(二硬脂酰磷脂酰乙醇胺)-PEG(聚乙二醇)(聚乙二醇分子量为2kDa)通过经典的薄膜水化法组成，其中质量比HSPC:CHO：DSPE-PEG＝56:39:2.5。HSPC、CHO溶解在氯仿中，随后蒸发浓缩干燥形成膜。氯仿完全蒸发后，加入1mL DSPE-PEG-NHS PBS溶液，然后在室温下以100W超声处理3min。然后将所得溶液分别通过800nm、400nm和200nm滤膜使DSPE-PEG-NHS插入脂质膜上，此过程应控制在10min内完成。
(3)POI识别基团NTZ抗体与PEG脂质杂化物质偶联：
偶联反应在冰水混合物环境中起始，并加以氮气保护。随后将脂质杂化物溶液添加到以800rpm(每分钟转数)搅拌的抗体PBS溶液中；然后搅拌反应在4℃，20rpm旋转器上孵育24小时。
(4)纯化：然后将反应混合物用50kDa离心超滤管超滤浓缩3次，随后使用PBS定容至100μL。蛋白质浓度由NanoDrop one C仪器通过IgG模式以A280处的吸光度确定，并通过BCA蛋白质定量试剂盒验证其结果。
实验结果：图47为NTZ-PEGlipo合成过程图。图48为MDA-MB-231(M231)人乳腺癌肿瘤细胞经500nM的NTZ-PEGlipo处理24小时后收蛋白进行蛋白电泳检测EGFR蛋白表达结果，结果显示NTZ-PEGlipo能够有效降解靶蛋白EGFR。
实施例10
参考表6，本实施例中，对于实施例10中的降解工具进行了制备，即POI识别基团与两亲性脂质连接臂偶联，随后与脂质杂化物质自组装。其中实施例10a和实施例10b中POI识别基团为：单抗药物尼妥珠(NTZ)；连接臂为带有NHS基团的NHS-PEG-DSPE。脂质杂化物质由膜骨架和胆固醇(CHO，cholesterol)组成，实施例10a中膜骨架为氢化大豆卵磷脂(HSPC)，其组成的降解工具为NTZ-lipo1，实施例10b中膜骨架为二硬脂酰磷脂酰胆碱(DSPC)，其组成的降解工具为NTZ-lipo2。实施例10c中POI识别基团为：HER2的治疗性单抗伊尼妥(INE，Inetetamab)，HER2为肿瘤增殖信号受体和标志物，连接臂为带有NHS基团的NHS-PEG-DSPE，脂质杂化物质由HSPC膜骨架和CHO组成，其组成的降解工具为INE-lipo。
实施例10d中POI识别基团为：小分子CDK4抑制剂Palbociclib(Palb),连接臂为带有NHS基团的NHS-PEG-DSPE，Palbociclib通过氨基与NHS反应进行偶联，脂质杂化物质由HSPC膜骨架和CHO组成，其组成的降解工具为Palb-lipo。
制备方法：
实验1：POI识别基团为抗体。其中，实施例10a和实施例10b中POI识别基团为：单抗药物尼妥珠(NTZ)。实施例10c中POI识别基团为：单抗药物伊尼妥(INE)。
(1)预处理：将原始抗体的缓冲液置换为磷酸盐缓冲液PBS。本实施例中，取NTZ、INE单抗溶液，用3kDa超滤管在4000×g离心2分钟，进而浓缩抗体，随后用PBS稀释；反复浓缩稀释3次后，缓冲液主要成分被置换为PBS，抗体溶液稀释后，通过NanoDrop one C仪器的模式以A280处的吸光度测定蛋白质浓度，并通过BCA蛋白质定量试剂 盒验证其结果。
(2)POI识别基团与两亲性脂质连接臂偶联反应：抗体与NHS-PEG(2kDa)-DSPE通过抗体上的氨基与DSPE(二硬脂酰磷脂酰乙醇胺)-PEG末端的NHS(N-羟基琥珀酰亚胺，N-Hydroxysuccinimide)基团反应。偶联反应在冰水混合物环境中起始，并加以氮气保护。为了提高分散性，可以选择将DSPE-PEG在PBS缓冲液中以40kHz超声处理3分钟，随后将等摩尔浓度和体积(摩尔比例不限于1:1，参考抗体偶联药物)的NHS-PEG-DSPE PBS溶液添加到以800rpm(每分钟转数)搅拌的抗体PBS溶液中；然后搅拌反应在4℃，20rpm旋转器上孵育24小时为了保持抗体活性更加倾向低转速和4度进行)。获得NTZ-PEG-DSPE反应混合物或INE-PEG-DSPE反应混合物。
其中，偶联方法包括但不限于非定点偶联和定点偶联，非定点偶联包括氨基偶联、羧基偶联、桥接巯基偶联。定点偶联如点击化学反应、硒键偶联、丝氨酸偶联、半胱氨酸偶联、非天然氨基酸偶联、酶催化后偶联、糖位点偶联。
(3)纯化：含有NTZ-PEG-DSPE或INE-PEG-DSPE的反应混合物用50kDa离心超滤管超滤浓缩3次，随后使用PBS定容至100μL。蛋白质浓度由NanoDrop one C仪器通过IgG模式以A280处的吸光度确定，并通过BCA蛋白质定量试剂盒验证其结果。
(4)脂质杂化物质制备：使用的脂质杂化物质为脂质体，由氢化大豆卵磷脂(HSPC)或二硬脂酰磷脂酰胆碱(DSPC)、胆固醇(CHO，cholesterol)、DSPE-PEG(2kDa)通过经典的薄膜水化法组成，其中质量比[HSPC或DSPC]:CHO：DSPE-PEG(不含POI识别基团)＝56:39:2.5。HSPC或DSPC、CHO溶解在氯仿中，随后蒸发浓缩干燥形成膜。
NTZ-lipo1,NTZ-lipo2,INE-lipo的制备：待前述脂质杂化物质溶液的溶剂氯仿完全蒸发后，加入1mL DSPE-PEG-抗体的PBS溶液，然后在室温下以100W超声处理3min。然后将所得溶液分别通过800nm、400nm和200nm滤膜。随后进一步纯化，将反应混合物用50kDa离心超滤管超滤浓缩3次，随后使用PBS定容至100μL。蛋白质浓度由NanoDrop one C仪器通过IgG模式以A280处的吸光度确定，并通过BCA蛋白质定量试剂盒验证其结果。
随后依据抗体浓度进行细胞实验。
实验2：POI识别基团为小分子。其中，实施例10d中，POI识别基团为Palbociclib小分子药物或β-amyloid探针AV-45。
Palbociclib为临床商用CDK4抑制剂,实施例中其脂质杂化物质的偶联物为Palb-lipo。CDK4为细胞周期蛋白依赖性激酶，广泛参与到细胞衰老肿瘤发生中。
Palbociclib与NHS-PEG(2kDa)-DSPE通过Palbociclib上的氨基与DSPE-PEG末端的NHS基团反应(摩尔比1.2:1)。在室温氮气保护下，搅拌反应24小时，获得Palb-PEG-DSPE反应混合物。Palb-PEG-DSPE透析24小时并冻干，通过紫外吸收峰的标准曲线确定浓度。
脂质杂化物质制备同本实施例的实验1。
Palb-lipo的制备，待前述脂质杂化物质溶液的溶剂氯仿完全蒸发后，加入1mL Palb-PEG-DSPE的PBS溶液，然后在室温下以100W超声处理3min。然后将所得溶液分别通过800nm、400nm和200nm滤膜。通过紫外吸收峰的标准曲线确定浓度，并依据Palb摩尔浓度进行细胞实验。
AV-45为阿尔兹海默症经典标志物β-amyloid探针，其脂质杂化物质偶联物为AV45-lipo。
β-amyloid为阿尔兹海默症的潜在致病蛋白，其为胞外蛋白，前体表达于细胞膜上，剪切后分泌至胞外形成寡聚体(β-amyloid 1-42oligomer)和纤维，其中寡聚体被认为毒性最大，作为胞外蛋白β-amyloid oligomer的积累带来神经炎症等病理风险，同位素标记的AV-45是一种临床商用β-amyloid探针。本实施例选用的为不含同位素的AV-45衍生物，AV-45-SH，AV-45具有蓝色自发荧光，便于细胞追踪，其巯基SH用于和Mal(马来酰亚胺)-PEG-DSPE偶联。
AV45-PEG-DSPE制备方法：将带有巯基的AV45-SH与Mal-PEG(分子量2kDa)-DSPE结合，即将AV45-SH与Mal-PEG-DSPE溶于DMSO，摩尔比1.2:1，避光氮气保护37℃水浴24h。随后透析冻干。通过通过紫外吸收峰的标准曲线确定浓度。
脂质杂化物质制备同本实施例实验1。AV45-lipo的制备：待前述脂质杂化物质溶液的溶剂氯仿完全蒸发后，加入1mL AV45-PEG-DSPE的PBS溶液，然后在室温下以100W超声处理3min。然后将所得溶液分别通过800nm、400nm和200nm滤膜。通过紫外吸收峰的标准曲线确定浓度，并依据AV45摩尔浓度进行细胞实验。
实验结果：图49为合成过程示意图。图50为M231人乳腺癌肿瘤细胞经不同浓度NTZ-lipo1处理24小时后收蛋白进行蛋白电泳检测EGFR蛋白表达结果，结果显示NTZ-lipo1能够有效降解靶蛋白EGFR。图51为脂质杂化物质由不同HSPC和CHO比例组成的NTZ-lipo1处理M231细胞24小时对靶蛋白EGFR降解效果的蛋白电泳结果，结果显示不同组成具有蛋白降解效果，而膜骨架HSPC和辅助脂质胆固醇的比例对蛋白降解效果有影响。图52为M231人乳腺癌肿瘤细胞经500nM NTZ-lipo2处理24小时后收蛋白进行蛋白电泳检测EGFR蛋白表达结果，结果显示NTZ-lipo2能够有效降解靶蛋白EGFR。图53为M231人乳腺癌肿瘤细胞经500nM INE-lipo处理24小时后收蛋白进行蛋白电泳检测HER2蛋白表达结果，结果显示INE-lipo能够有效降解靶蛋白。
图54为Palb-lipo所用脂质连接臂与POI识别基团偶联后的Palb-PEG-DSPE的核磁共振氢谱(1H NMR)，结果显示成功连接。图65为AV45-lipo所用脂质连接臂与POI识别基团偶联后的AV45-PEG-DSPE的核磁共振氢谱，结果显示成功连接。图56为M231人乳腺癌肿瘤细胞经不同浓度Palb-lipo处理24小时后收蛋白进行蛋白电泳检测CDK4蛋白表达结果，结果显示Palb-lipo能够有效降解靶蛋白。图57为AV45-lipo结构示意图以及AV45-SH结构。图58为人胶质细胞经不同浓度AV45-lipo处理24小时后进行共聚焦显微镜成像，即检测β-amyloid(Aβ)被劫持进入细胞效果。10μM的FITC绿色染料标记的β-amyloid 1-42多肽制备的寡聚体与10μM的AV45-lipo共孵育24小时，随后通过PBS洗掉未进入细胞的处理物，进行共聚焦显微镜成像，检测β-amyloid 1-42寡聚体的绿色荧光，绿色荧光强则表明被劫持进入细胞量更多，理论上更多的劫持和吞噬以及溶酶体转移能带来更好的降解，以减轻胞外β-amyloid oligomer的积累带毒性，溶酶体染料LysoTracker用来标记溶酶体，比例尺为10μm。
实施例11
参考表6，本实施例中，对于实施例11中的降解工具进行了制备，即POI识别基团与两亲性脂质连接臂偶联，随后与脂质杂化物质自组装。其中POI识别基团为：单抗药物尼妥珠(NTZ)；连接臂为带有NHS基团的NHS-PEG-DSPE或 NHS-PEG-DMG(1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇,聚乙二醇末端修饰N-羟基琥珀酰亚胺)。脂质杂化物质由膜骨架HSPC、胆固醇，以及阳离子脂质DOTAP(溴化三甲基-2,3-二油酰氧基丙基铵)组成。阳离子脂质DOTAP是一种代表性的阳离子脂质，阳离子脂质有助于脂质杂化物质形成Lipid nanoparticle(LNP)用于包载带有负电的核酸类药物。当脂质杂化物质的组成为PEG-DSPE/胆固醇/HSPC，其组成的降解工具为NTZ-LNP1,当NTZ-LNP1载有无义序列空载小干扰RNA(siRNA)其组成的降解工具为NTZ-LNP1s；当脂质杂化物质的组成为PEG-DMG/胆固醇/HSPC，其组成的降解工具为NTZ-LNP2。
制备方法：
其预处理、POI识别基团和连接臂的连接、纯化，采用实施例10中所用方法。
脂质杂化物质制备：使用的脂质杂化物质为脂质纳米颗粒，由氢化大豆卵磷脂(HSPC)或二硬脂酰磷脂酰胆碱(DSPC)、胆固醇(CHO，cholesterol)、DSPE-PEG(2kDa)通过经典的薄膜水化法组成，其中质量比[HSPC或DSPC]:CHO：DSPE-PEG(不含POI识别基团)＝56:39:2.5。HSPC、CHO溶解在氯仿中，随后蒸发浓缩干燥形成膜。
NTZ-LNP1:上述脂质杂化物合成后，待氯仿完全蒸发，加入1mL DSPE-PEG-抗体的PBS溶液，同时加入DOTAP溶液(摩尔比HSPC:DOTAP＝1:1)然后在室温下以100W超声处理3min。然后将所得溶液分别通过800nm、400nm和200nm滤膜。
NTZ-LNP1s：上述脂质杂化物合成后，待氯仿完全蒸发，加入1mL DSPE-PEG-抗体的PBS溶液，siRNA的量根据与DOTAP的氮磷比(N/P＝3:1)计算。同时加入DOTAP溶液(摩尔比HSPC:DOTAP＝1:1)与siRNA溶液。然后在室温下以100W超声处理3min。然后将所得溶液分别通过800nm、400nm和200nm滤膜。
纯化：然后将反应混合物用50kDa离心超滤管超滤浓缩3次，随后使用PBS定容至100μL。蛋白质浓度由NanoDrop one C仪器通过IgG模式以A280处的吸光度确定，并通过BCA蛋白质定量试剂盒验证其结果。
实验结果：图59为NTZ-LNP1、NTZ-LNP1s、NTZ-LNP2结构示意图。图60为所述脂质杂化物质的动态光散射(DLS)检测的粒径分布。图61为M231人乳腺癌肿瘤细胞经500nM NTZ-LNP1处理24小时后收蛋白进行蛋白电泳检测EGFR蛋白表达结果，其中实施例10中的lipo1也一同作为对照。结果显示NTZ-LNP1能够有效降解靶蛋白EGFR。图62为M231人乳腺癌肿瘤细胞经NTZ-LNP1s处理24小时后收蛋白进行蛋白电泳检测EGFR蛋白表达结果，结果显示NTZ-LNP1s能够有效降解靶蛋白EGFR。图63为M231人乳腺癌肿瘤细胞经NTZ-LNP2处理24小时后收蛋白进行蛋白电泳检测EGFR蛋白表达结果，结果显示NTZ-LNP2能够有效降解靶蛋白EGFR。
实施例12
参考表6，本实施例中，对于实施例12中的降解工具进行了制备，即POI识别基团与两亲性脂质连接臂偶联，随后与脂质杂化物质自组装。其中POI识别基团为：单抗药物尼妥珠(NTZ)；连接臂为带有NHS基团的NHS-PEG-DSPE。脂质杂化物质由外泌体(exosome)组成。外泌体是由真核生物如动物或植物细胞分泌的细胞外囊泡，是自发形成的，可以进行细胞间通讯，通常含有蛋白和少量核酸，外泌体具有较高的生物相容性和载药能力，其表面为磷脂膜结构。
制备方法：
其预处理、POI识别基团和连接臂的连接、纯化，采用实施例10中所用方法。
脂质杂化物质制备：外泌体由DC2.4细胞根据标准步骤分离并纯化鉴定，外泌体通过超声和挤膜两种方式进行连接臂的偶联。即外泌体PBS溶液加入1mL DSPE-PEG-抗体的PBS溶液，
连接臂和脂质杂化物质的结合采用挤膜或超声法。超声法即在外泌体的PBS溶液中加入DSPE-PEG-抗体的PBS溶液(例如在1mL体系中质量比外泌体：DSPE-PEG＝1mg：25μg，即40：1)，超声处理30秒，然后在37℃下孵育1小时。挤膜法即：在外泌体中加入1mL DSPE-PEG-抗体的PBS溶液(质量比外泌体：DSPE-PEG＝1mg：25μg，即40：1)然后在室温下以100W超声处理3min。然后将所得溶液分别通过800nm、400nm和200nm滤膜。
纯化：然后将反应混合物用50kDa离心超滤管超滤浓缩3次，随后使用PBS定容至100μL。蛋白质浓度由NanoDrop one C仪器通过IgG模式以A280处的吸光度确定，并通过BCA蛋白质定量试剂盒验证其结果。
实验结果：图64为所述脂质杂化物质的DLS检测的粒径和平均分散系数PDI(Polymer dispersity index)，PDI越小，粒径约均匀，结果表示粒径均匀。图65为M231人乳腺癌肿瘤细胞经不同浓度NTZ-exo处理24小时后收蛋白进行蛋白电泳检测EGFR蛋白表达结果，结果显示NTZ-exo能够有效降解靶蛋白EGFR。
实施例13
参考表6，本实施例中，对于实施例13中的降解工具进行了制备，即脂质杂化物质包覆于纳米颗粒表面，其POI识别基团与两亲性脂质连接臂偶联，随后与脂质杂化物质自组装，POI识别基团暴露于外部。其中POI识别基团为：靶向EGFR的单抗药物尼妥珠(NTZ)或西妥昔(CTX)；连接臂为带有NHS基团的NHS-PEG-DSPE。脂质杂化物质由HSPC/胆固醇组成并包裹PLGA纳米颗粒时，该降解工具为NTZ-lipoP。聚乳酸-羟基乙酸共聚物(PLGA)为具有代表性的聚合物纳米颗粒，其具有疏水性，具有较高的生物相容性，性质稳定并可以被生物体降解，被广泛用于医疗。脂质杂化物质为小鼠红细胞膜(RBCm)组成并包裹缩醛化葡聚糖(Dextran),POI识别基团为CTX组成时，该降解工具为CTX-RBCmD。葡聚糖为具有代表性的亲水生物可降解纳米颗粒。红细胞膜是细胞膜和细胞器膜的代表，是一种脂质杂化物质，便于分离提取。
制备方法：
其预处理、POI识别基团和连接臂的连接、纯化，采用实施例10中所用方法。
脂质杂化物质制备：
NTZ-lipoP制备：由HSPC、CHO通过经典的薄膜复水法组成，其中各化合物按照质量比HSPC:CHO：PEG-DSPE(不含POI识别基团)＝56:39:2.5。HSPC与CHO溶解在氯仿中，随后浓缩蒸发形成脂质膜。氯仿完全蒸发后，加入1mL溶解在PBS中的抗体-PEG-DSPE重悬，然后在室温下以100W超声处理3min。然后将所得溶液分别通过800nm、400nm和200nm滤膜使DSPE-PEG-抗体插入脂质膜上。
包裹PLGA：PLGA(15kDa)溶解在DMF中(10μg/μL),每10秒加2μL加入1mL PBS溶液中，700rpm搅拌3h，待 DMF挥发后每0.01μmol的PLGA外边裹1mg的LIPO(指的是HSPC+CHO共1mg)LIPO适量的HSPC和CHO溶在氯仿中，蒸发浓缩后用DSPE-PEG-NTZ的PBS溶液重悬，100W超声3min。37℃摇床共孵育30min，室温100W超声5min，过800，400，200nm的膜。两者混合物再与PLGA的PBS溶液混合，100W冰浴超声2min，过800，400，200nm的膜，超滤浓缩。IgG测浓度。
CTX-RBCmD制备：CTX-PEG-DSPE合成方法如实施例10。
将1mg缩醛化葡聚糖溶于200μL的四氢呋喃中，每滴10μL，每10s一滴滴入搅拌中的1mL pH＝8的碱水中，700rpm搅动3小时。取小鼠红细胞膜1mg重悬于1mL的PBS中。红细胞膜的PBS溶液与CTX-PEG-DSPE的PEG溶液(根据膜：缩醛化葡聚糖＝1mg:1mg计算，缩醛化葡聚糖核心与DSPE-PEG的摩尔比为10:1)混合后200rpm 37℃摇床孵育30min，随后100W室温超声5min，然后将所得溶液依次通过400nm和200nm滤膜过滤，以获得均匀的尺寸。然后与缩醛化葡聚糖溶液混合，100W冰浴超声2min，将所得溶液分别通过400nm和200nm滤膜过滤以获得目的产物。然后进行超滤浓缩。通过NanoDrop one C(Thermofisher)以IgG模式以A280处的吸光度测定蛋白质浓度，并通过BCA蛋白质定量试剂盒验证其结果。
表6、NTZ-lipoP和未经NTZ-PEG-DSPE连接的lipo-PLGA的DLS粒径统计和PDI统计结果
实验结果：图66为NTZ-lipoP结构示意图。表6为NTZ-lipoP和未经NTZ-PEG-DSPE连接的lipo-PLGA的DLS粒径统计和PDI统计结果，结果显示粒径均一，连接NTZ-PEG-DSPE后水合粒径略微增大。图67为M231人乳腺癌肿瘤细胞经不同浓度NTZ-lipoP处理24小时后收蛋白进行蛋白电泳检测EGFR蛋白表达结果，结果显示其能够有效降解靶蛋白EGFR。图68为CTX-RBCmD结构示意图。图69为DLS粒径分布结果图。图70为M231人乳腺癌肿瘤细胞经500nM CTX-RBCmD处理24小时后收蛋白进行蛋白电泳检测EGFR蛋白表达结果，结果显示其能够有效降解靶蛋白EGFR。
工业实用性
本公开通过将POI识别基团修饰于纳米颗粒的表面，或POI识别基团连接于连接臂，或POI识别基团修饰于连接臂并通过连接臂连接于纳米颗粒表面，POI识别基团组装后暴露于纳米颗粒或连接臂的外部，其中，纳米颗粒(NP,Nanoparticle)能够不依赖特殊设计的受体-配体匹配对进行细胞渗透，并且可以偶联小分子、多肽、核酸适配体、抗体等POI识别基团，特异性地进行蛋白质劫持和靶向降解，从而实现基于纳米颗粒的TPD工具(TPD-NP)的组装。这种便捷的基于纳米颗粒的TPD工具极易获得载药和组织特异性的靶向的能力，使得药物和蛋白降解联合治疗以及转化/精准医学成为可能。TPD-NP的发明和其机制的探索，大大扩展了TPD工具的范围，并为TPD和纳米递送领域提供了基础知识，并且原则上可以在体内降解多种人类疾病相关的细胞外/膜相关/细胞内蛋白。
本公开首次系统地提出纳米颗粒介导的蛋白质降解，对于TPD和纳米递送提供了新的途径。本公开的纳米蛋白降解工具结构灵活，便于改造，可获得载药、靶向、穿越生物屏障的能力。本公开的纳米蛋白降解工具具有普适性，靶点可随意更换，三种组分均可以替换，应用场景广。本公开纳米蛋白降解工具所有组分可以是临床批准的材料，体内应用潜力高，有转化价值，本公开的纳米蛋白降解工具无须从头合成，为即用型平台，大大降低了开发和生产的复杂性和难度。相比PROTAC降解胞内蛋白，LYTAC降解胞外/膜蛋白，TPD-NP可以降解胞外/胞内/膜蛋白。另外，相对于LYTAC等胞外/膜蛋白降解工具，TPD-NP不需要额外设计辅助所劫持蛋白被内吞的结构。相比目前已有的TPD工具，本公开的纳米蛋白降解工具不需要特殊结构引导蛋白降解。纳米颗粒可以载药、可以设计成为可控释放，可以设计成为光热磁等协同治疗材料，可以成像、造影进而进行蛋白降解的协同治疗和诊疗一体化。NP的体液稳定性可以减少药物损失，提高成药潜力。
此外，本公开通过将POI识别基团修饰于脂质杂化物质的表面，并且POI识别基团组装后暴露于脂质杂化物质的外部，实现了基于脂质杂化物质的靶蛋白降解，使得构建蛋白降解工具的合成难度极大降低，通过“即插即用”模式，使得海量与靶蛋白具有结合力的化合物、多肽、抗体和核酸适配体等能够升级成为蛋白降解类药物，进而在传统脂质体(Liposome)、脂质纳米颗粒(LNP)相关领域，如mRNA疫苗、核酸递送载体、药物递送中发挥新的功能，从而实现在科研和工业应用中进行联合疗法开发。
本公开中所采取的即插即用式的组合方式极为便捷方便、并且结构可以根据需要灵活组装，组装材料可以完全来源于临床易于接受的材料，并且创造性地赋予了纳米载体递送性能以外的蛋白降解功能，也扩宽了生物技术药物的范畴。
而且，配体靶向的脂质类纳米颗粒(脂质杂化物质)作为蛋白降解工具的应用和其机制的探索尚属空白，该发明大大扩展了目前脂质类纳米颗粒的应用范围，并为TPD和纳米递送领域提供了基础知识，并且原则上可以在体内降解多种人类疾病相关的细胞外/膜相关/细胞内蛋白。
以上所述的是本公开的可选实施方式和相应实施例，应当指出，对于本领域的普通技术人员来说，在不脱离本公开创造构思的前提，还可以做出若干变形和改进，包括但不限于比例、流程、用量的调整，这些都属于本公开的保护范围之内。

Claims (17)

  1. 一种纳米蛋白降解工具，其特征在于，包括：第一降解工具、第二降解工具和第三降解工具中的一种或多种组合；
    其中，所述第一降解工具由POI识别基团与连接臂连接构成；所述第二降解工具由所述POI识别基团与纳米颗粒连接构成；所述第三降解工具由所述POI识别基团通过所述连接臂与所述纳米颗粒连接构成；
    所述POI识别基团包括能与POI特异性结合的抗体、蛋白、多肽、核酸适配体或小分子。
  2. 如权利要求1所述纳米蛋白降解工具，其特征在于，
    所述第一降解工具中，所述POI识别基团与所述连接臂构成一组连接单元；所述第一降解工具是由包括多组设于核心处的所述连接臂以及与所述连接臂连接的、设于外围的所述POI识别基团的所述连接单元所组成的具有多层结构的所述纳米蛋白降解工具；
    所述第二降解工具是由包括设于核心处的所述纳米颗粒以及与所述纳米颗粒连接的、设于外围的多个所述POI识别基团的连接单元所组成的具有多层结构的所述纳米蛋白降解工具；
    所述第三降解工具中，所述POI识别基团通过所述连接臂与所述纳米颗粒连接；所述第三降解工具是由包括设于核心处的所述纳米颗粒，与所述纳米颗粒连接的、设于中间层的多组所述连接臂以及设于外围的、与所述连接臂连接的所述POI识别基团的连接单元所组成的具有多层结构的所述纳米蛋白降解工具。
  3. 如权利要求1所述纳米蛋白降解工具，其特征在于，
    所述连接臂包括亲水聚合物、疏水聚合物和两亲性聚合物；
    所述连接臂的分子量为0-1000kDa；
    所述两亲性聚合物包括：两亲性嵌段聚合物，所述亲水聚合物与疏水小分子组成的两亲性聚合物，以及所述疏水聚合物与亲水小分子组成的两亲性聚合物。
  4. 如权利要求3所述纳米蛋白降解工具，其特征在于，所述两亲性聚合物为链状或支化分子结构的聚合物，其至少存在一个具有亲水性的分子端，以及一个具有疏水性的分子端。
  5. 如权利要求4所述纳米蛋白降解工具，所述两亲性聚合物为直链分子结构，其一端为亲水性的分子端，另一端为具有疏水性的分子端。
  6. 如权利要求3所述纳米蛋白降解工具，其特征在于，所述纳米颗粒包括表面单一纳米颗粒和杂化纳米颗粒；
    所述表面单一纳米颗粒包括亲水性颗粒、疏水性颗粒和无机纳米颗粒；
    所述杂化纳米颗粒为通过杂化物质对所述表面单一纳米颗粒改性后的杂化纳米颗粒；
    其中，所述杂化物质为改性膜；所述改性膜包括细胞膜、外泌体、油膜、水凝胶和脂质体；
    所述杂化纳米颗粒为通过所述改性膜包覆于所述表面单一纳米颗粒外表面构成的颗粒，以便于所述表面单一纳米颗粒在改性成为所述杂化纳米颗粒后，能与所述亲水聚合物的臂、疏水聚合物的臂、或所述POI识别基团相连接；
    所述纳米颗粒的粒径为5-1000nm。
  7. 如权利要求6所述纳米蛋白降解工具，其特征在于，
    所述第三降解工具中，所述纳米颗粒为疏水性颗粒、亲水性颗粒或所述表面单一纳米颗粒外包覆有所述改性膜，且所述连接臂为两亲性聚合物、亲水聚合物或者疏水聚合物时，所述连接臂与所述纳米颗粒的连接方式包括：所述连接臂通过非共价键合于所述纳米颗粒上，或者通过所述连接臂上修饰的活性基团共价键合于所述纳米颗粒上。
  8. 如权利要求1所述纳米蛋白降解工具，其特征在于，
    所述POI识别基团中的抗体为治疗性单克隆抗体、多特异性抗体、纳米抗体及前述抗体衍生物或抗体偶联药物；
    所述多肽为具有POI特异性结合能力的多肽；
    所述小分子为具有POI特异性结合能力的小分子化合物。
  9. 一种治疗和预防蛋白异常积累疾病的方法，包括向受试者施用治疗有效量的如权利要求1所述的纳米蛋白降解工具，其中，所述蛋白异常积累疾病包括肿瘤、免疫系统疾病、炎症和病原体感染、神经退行性疾病、血液系统疾病和代谢类疾病。
  10. 一种基于脂质的蛋白降解工具，其特征在于，包括：
    POI识别基团，以及与所述POI识别基团连接的脂质杂化物质；
    其中，所述POI识别基团包括能与POI特异性结合的抗体、蛋白、多肽、核酸适配体或小分子；
    所述脂质杂化物质包括脂质体、外泌体、细胞膜以及LNP。
  11. 如权利要求10所述基于脂质的蛋白降解工具，其特征在于，
    在所述POI识别基团与所述脂质杂化物质偶联时，所述基于脂质的蛋白降解工具是由包括处于核心的所述脂质杂化物质与设于外围的所述POI识别基团组成的用于蛋白降解的纳米颗粒。
  12. 如权利要求10所述基于脂质的蛋白降解工具，其特征在于，
    所述基于脂质的蛋白降解工具还包括，在所述POI识别基团与所述脂质杂化物质之间设有连接构件的所述基于脂质的蛋白降解工具；
    所述连接构件的分子量为0-1000kDa；
    所述连接构件为聚合物连接臂和脂质连接臂中的一种；
    所述聚合物连接臂包括亲水聚合物、疏水聚合物和两亲性聚合物；
    所述脂质连接臂为两亲性的脂质连接臂。
  13. 如权利要求12所述基于脂质的蛋白降解工具，其特征在于，在所述POI识别基团通过所述连接构件与所述脂质杂化物质偶联时，所述POI识别基团与所述连接构件构成一组连接单元；所述基于脂质的蛋白降解工具是由包括设于核心处的所述脂质杂化物质，设于中间层的、与所述脂质杂化物质连接的多组所述连接构件以及设于外围的、与所述连接构件连接的所述POI识别基团的多组连接单元所组成的具有多层结构的所述基于脂质的蛋白降解工具。
  14. 如权利要求12所述基于脂质的蛋白降解工具，其特征在于，所述脂质连接臂中至少包括两端，一端为能与所述脂质杂化物质相连接的亲脂末端，另一端为亲水末端；
    亲脂末端为脂质分子。
  15. 如权利要求12所述基于脂质的蛋白降解工具，其特征在于，
    所述两亲性聚合物为链状或支化分子结构的聚合物，其至少存在一个具有亲水性的分子端，以及一个具有疏水性的分子端。
  16. 如权利要求15所述纳米蛋白降解工具，所述两亲性聚合物为直链分子结构，其一端为亲水性的分子端，另一端为具有疏水性的分子端。
  17. 如权利要求10所述基于脂质的蛋白降解工具，其特征在于，还包括纳米颗粒，其中，所述纳米颗粒被所述脂质杂化物质包覆于所述基于脂质的蛋白降解工具的核心处；
    所述纳米颗粒包括亲水性颗粒、疏水性颗粒和无机纳米颗粒；
    所述纳米颗粒的粒径为5-1000nm。
PCT/CN2023/104199 2022-07-29 2023-06-29 一种纳米蛋白降解工具、应用及其制备方法以及一种基于脂质的蛋白降解工具、应用及其制备方法 WO2024022009A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202210906564.6 2022-07-29
CN202210911853.5 2022-07-29
CN202210911853.5A CN115260304A (zh) 2022-07-29 2022-07-29 一种基于脂质的蛋白降解工具、应用及其制备方法
CN202210906564.6A CN115043932A (zh) 2022-07-29 2022-07-29 一种纳米蛋白降解工具、应用及其制备方法

Publications (1)

Publication Number Publication Date
WO2024022009A1 true WO2024022009A1 (zh) 2024-02-01

Family

ID=89665492

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/104199 WO2024022009A1 (zh) 2022-07-29 2023-06-29 一种纳米蛋白降解工具、应用及其制备方法以及一种基于脂质的蛋白降解工具、应用及其制备方法

Country Status (2)

Country Link
US (1) US20240033374A1 (zh)
WO (1) WO2024022009A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010126319A2 (ko) * 2009-04-30 2010-11-04 고려대학교 산학협력단 항체의 Ｆｃ 영역에 특이적 결합능을 가지는 리포펩타이드 및 그를 포함하는 항원 인지형 지질 나노입자
CN111214461A (zh) * 2020-03-24 2020-06-02 河南大学 糖靶向修饰siRNA纳米粒子的制备及应用
WO2022036318A1 (en) * 2020-08-14 2022-02-17 Case Western Reserve University Plasmid vectors and nanoparticles for treating ocular disorders
CN115043932A (zh) * 2022-07-29 2022-09-13 河南大学 一种纳米蛋白降解工具、应用及其制备方法
CN115260304A (zh) * 2022-07-29 2022-11-01 河南大学 一种基于脂质的蛋白降解工具、应用及其制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010126319A2 (ko) * 2009-04-30 2010-11-04 고려대학교 산학협력단 항체의 Ｆｃ 영역에 특이적 결합능을 가지는 리포펩타이드 및 그를 포함하는 항원 인지형 지질 나노입자
CN111214461A (zh) * 2020-03-24 2020-06-02 河南大学 糖靶向修饰siRNA纳米粒子的制备及应用
WO2022036318A1 (en) * 2020-08-14 2022-02-17 Case Western Reserve University Plasmid vectors and nanoparticles for treating ocular disorders
CN115043932A (zh) * 2022-07-29 2022-09-13 河南大学 一种纳米蛋白降解工具、应用及其制备方法
CN115260304A (zh) * 2022-07-29 2022-11-01 河南大学 一种基于脂质的蛋白降解工具、应用及其制备方法

Also Published As

Publication number Publication date
US20240033374A1 (en) 2024-02-01

Similar Documents

Publication Publication Date Title
US11406597B2 (en) Fusogenic liposome-coated porous silicon nanoparticles
US20150272885A1 (en) Porous nanoparticle-supported lipid bilayers (protocells) for targeted delivery including transdermal delivery of cargo and methods thereof
Tang et al. Soft materials as biological and artificial membranes
CN109803687A (zh) 纳米载体向肿瘤的抗体介导的自催化靶向递送
Menon et al. Fabrication of active targeting lipid nanoparticles: Challenges and perspectives
Gong et al. Enzyme-induced transformable peptide nanocarriers with enhanced drug permeability and retention to improve tumor nanotherapy efficacy
US20130243867A1 (en) Micelle compositions and methods for their use
Ghafary et al. Design and preparation of a theranostic peptideticle for targeted cancer therapy: Peptide-based codelivery of doxorubicin/curcumin and graphene quantum dots
Tang et al. Synergistic targeted delivery of payload into tumor cells by dual-ligand liposomes co-modified with cholesterol anchored transferrin and TAT
US20150050330A1 (en) Compositions and methods for polymer-caged liposomes
Wang et al. Biomimetic exosomes: a new generation of drug delivery system
Qu et al. Targeted delivery of doxorubicin via CD147-mediated ROS/pH dual-sensitive nanomicelles for the efficient therapy of hepatocellular carcinoma
US11730820B2 (en) Peptidic blocks for nucleic acid delivery
Goh et al. nCVTs: a hybrid smart tumour targeting platform
Kato et al. Synthesis and evaluation of a novel adapter lipid derivative for preparation of cyclic peptide-modified PEGylated liposomes: Application of cyclic RGD peptide
Chen et al. Dual-pH sensitive charge-reversal drug delivery system for highly precise and penetrative chemotherapy
CN115043932A (zh) 一种纳米蛋白降解工具、应用及其制备方法
CN106913880B (zh) 一种含有rspo1的靶向给药系统及其制备与应用
JP2003514843A (ja) モジュラー標的化リポソーム送達システム
CN115260304A (zh) 一种基于脂质的蛋白降解工具、应用及其制备方法
WO2024022009A1 (zh) 一种纳米蛋白降解工具、应用及其制备方法以及一种基于脂质的蛋白降解工具、应用及其制备方法
US9326938B2 (en) Oligomer-contained nanoparticle complex release system
KR102475540B1 (ko) 약물이 담지된 지질 나노입자 제조 방법 및 제조 장치
Misra et al. Functionalized liposomes: A nanovesicular system
Khaleghi et al. Anti-HER2 VHH targeted fluorescent liposome as bimodal nanoparticle for drug delivery and optical imaging

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23845222

Country of ref document: EP

Kind code of ref document: A1