WO2019061648A1 - Polypeptide ciblant le sulfate a de chondroïtine placentaire, système d'administration ciblée, son procédé de préparation et son application - Google Patents

Polypeptide ciblant le sulfate a de chondroïtine placentaire, système d'administration ciblée, son procédé de préparation et son application Download PDF

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WO2019061648A1
WO2019061648A1 PCT/CN2017/108646 CN2017108646W WO2019061648A1 WO 2019061648 A1 WO2019061648 A1 WO 2019061648A1 CN 2017108646 W CN2017108646 W CN 2017108646W WO 2019061648 A1 WO2019061648 A1 WO 2019061648A1
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delivery system
targeted delivery
polypeptide
drug
targeted
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PCT/CN2017/108646
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English (en)
Chinese (zh)
Inventor
范秀军
张保珍
谭仑波
汪宝蓓
程国钢
肖仲琳
韩金雨
陈指龙
肖天霞
李梦霞
郑明彬
蔡林涛
张键
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中国科学院深圳先进技术研究院
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Priority claimed from CN201710905200.5A external-priority patent/CN109568598B/zh
Priority claimed from CN201710905199.6A external-priority patent/CN109589416B/zh
Priority claimed from CN201710906204.5A external-priority patent/CN109568596B/zh
Priority claimed from CN201710906587.6A external-priority patent/CN109589413B/zh
Priority claimed from CN201710903483.XA external-priority patent/CN109568289B/zh
Priority claimed from CN201710906586.1A external-priority patent/CN109568597B/zh
Priority claimed from CN201710903031.1A external-priority patent/CN109568268A/zh
Application filed by 中国科学院深圳先进技术研究院 filed Critical 中国科学院深圳先进技术研究院
Publication of WO2019061648A1 publication Critical patent/WO2019061648A1/fr

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    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the invention relates to the technical field of medicines, in particular to a polypeptide targeting a placenta-like chondroitin sulfate A, a targeted delivery system, a preparation method and application thereof.
  • Chondroitin sulfate is a class of glycosaminoglycans covalently linked to form proteoglycans on proteins. Chondroitin sulfate is widely distributed in the extracellular matrix and cell surface of animal tissues and plays an important physiological function. Although the polysaccharide skeleton of chondroitin sulfate is simple, there is a large difference in the degree of sulfation, the distribution of sulfate groups, and the distribution of two diisomeric uronic acids in the chain. The fine structure of chondroitin sulfate determines its functional specificity and interaction with a variety of protein molecules.
  • Placenta-like chondroitin sulfate A belongs to the family of glycosaminoglycans, which are linear polymers of alternating amino sugars and hexuronic acid residues attached to proteoglycans, with glycosylation patterns and conventional Chondroitin sulfate is different.
  • pl-CSA is the cause of red blood cell isolation of Plasmodium infection in the placenta; in 2015, Ali Salanti et al. Targeting Human Cancer by a Glycosaminoglycan Binding Malaria Protein states that pl-CSA is expressed in a variety of cancer cells, indicating a new direction for the treatment of cancer.
  • the specific receptor for pl-CSA is unknown, and there are no reports on receptors, delivery systems, etc. that specifically bind to pl-CSA.
  • the present invention provides a polypeptide targeting a placenta-like chondroitin sulfate A, a targeted delivery system, a preparation method and application thereof, and a direction for the diagnosis and treatment of a disease associated with placenta-like chondroitin sulfate A. .
  • the present invention provides a polypeptide which targets placenta-like chondroitin sulfate A (pl-CSA), wherein the amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: Amino acid sequence.
  • pl-CSA placenta-like chondroitin sulfate A
  • the polypeptide may be a sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2 or as SEQ ID NO: 3, or in the sequence set forth in SEQ ID NO: 1 - SEQ ID NO: A variety.
  • the polypeptide is highly targeted to pl-CSA.
  • the polypeptide can be modified on a common drug carrier or gene carrier such as a polymer (such as polyethyleneimine, chitosan, etc.), liposome, gold nanoparticle, silica, serum albumin, etc. to obtain a targeted placenta.
  • a targeted delivery system for chondroitin sulfate A can also be coupled to a small molecule drug to provide a targeted delivery system that is specifically a polypeptide drug conjugate.
  • the invention provides a targeted delivery system that targets placenta-like chondroitin sulfate A, the targeted delivery system comprising the polypeptide that targets pl-CSA.
  • the targeted delivery system further comprises: a core, a single layer of lipid molecular layer encapsulating the inner core, and a shell targeting pl-CSA; the kernel comprising the a hydrophobic polymer, wherein the outer shell is an amphiphilic macromolecule grafted to the polypeptide, and a hydrophobic end of the amphiphilic macromolecule is interspersed in the single layer lipid molecular layer, the amphipathic
  • the hydrophilic end of the molecule is linked to the polypeptide that targets pl-CSA by an amide bond that is exposed outside of the monolayer of lipid molecules.
  • the targeted delivery system is spherical and has a diameter of nanometer. It is preferably 80-150 nm. The particle size is measured using a transmission electron microscope.
  • the nano-scale spheroidal delivery system helps to reduce renal excretion clearance, reticuloendothelial system absorption and phagocytic cell recognition; secondly, it can smoothly reach the target tissue through the capillary endothelial cell gap.
  • the targeted delivery system further comprises a target delivery material loaded in the hydrophobic polymer, the target delivery material and the hydrophobic polymer forming the core together.
  • a target delivery material loaded in the hydrophobic polymer, the target delivery material and the hydrophobic polymer forming the core together.
  • the hydrophobic polymer can adsorb or wrap the target delivery.
  • the target delivery object includes at least one of a contrast agent, a fluorescence tracer, a photothermal conversion reagent, a pregnancy drug, and an antitumor drug, but is not limited thereto.
  • the targeted delivery system may be referred to as a "targeted delivery nanobubble", at which point the hydrophobic polymer encapsulates the target delivery.
  • the mass ratio of the hydrophobic polymer, the monolayer lipid molecule, and the amphiphilic macromolecule is 1: (0.04-0.3) (0.1-0.6).
  • a spherical structure having a relatively regular morphology, a uniform particle size distribution, and good dispersibility can be formed between the components of the placenta-targeted delivery system, and the structure of the placenta-targeted delivery system is stable. It is not easy to produce repulsion in the blood, which is beneficial to quickly reach the target tissue expressing pl-CSA.
  • the mass ratio of the hydrophobic polymer to the amphiphilic macromolecule is 1: (0.05-0.3), 1: (0.04-0.2), or 1: (0.05-0.2).
  • the mass ratio of the hydrophobic polymer to the amphiphilic macromolecule is 1: (0.1-0.4), 1: (0.2-0.6), or 1: (0.2-0.4).
  • the mass ratio of the hydrophobic polymer to the target delivery material is 1: (0.1-0.8).
  • it can be 1:0.2, 1:0.25, 1:0.3, 1:0.5 or 1:0.6.
  • the mass ratio of the hydrophobic polymer to the target delivery material is 1: (0.25-0.75), 1: (0.1-0.6), 1: (0.3-0.5) or 1: (0.1-0.2) ), even 1: (0.1-0.16).
  • the target delivery contains an antitumor drug
  • the mass ratio of the hydrophobic polymer to the target delivery is 1: (0.25-0.75).
  • the target delivery material contains both an anti-tumor drug and a fluorescence tracer, or both an anti-tumor drug and a contrast agent. At this time, the mass ratio of the antitumor drug to the fluorescent tracer or contrast agent is 1:1.
  • the mass ratio of the hydrophobic polymer to the target delivery is 1: (0.2-0.6).
  • the target delivery contains a pregnancy drug and an ultrasound contrast agent.
  • the mass ratio of the pregnancy drug to the ultrasound contrast agent is 1: (0.1-4).
  • the mass ratio of the pregnancy drug to the ultrasound contrast agent is 1: (1-4), more preferably 1:1.
  • the amphiphilic macromolecule has a hydrophobic end and a hydrophilic end attached to the lipid end.
  • the hydrophobic end of the amphiphilic macromolecule can facilitate insertion of the amphiphilic macromolecule into the monolayer lipid molecular layer, and the hydrophilic end is grafted with the polypeptide and extends in the nanobubble The outside.
  • the mass ratio of the amphiphilic macromolecule to the polypeptide is 1:1-4. At this mass ratio, the grafting ratio of the polypeptide to the amphiphilic macromolecule is higher.
  • the hydrophobic polymer can adsorb or wrap the target delivery material, which together constitute a hydrophobic core; the single layer lipid molecules can be self-assembled into a single Lipid molecule And encapsulating the hydrophobic core, the hydrophobic end of the amphiphilic macromolecular compound is physically intercalated with the lipid molecule in the monolayer lipid molecular layer to interpenetrate the single layer lipid molecule
  • the polypeptide is covalently linked to the hydrophilic end of the amphiphilic macromolecular compound and extends outside of the targeted delivery system, providing a hydrophilic shell and targeting for the targeted delivery system
  • the receptor of pl-CSA therefore, the targeted delivery system has better targeting to tissues that are inappropriately expressed by pl-CSA, such as placental trophoblast tissue, tumor tissue.
  • the targeted delivery system comprises: a hydrophobic polymer layer, a viscous molecule, and an outer shell, wherein the viscous molecule adheres to the hydrophobicity a surface of the polymer layer, the outer shell being an amphiphilic macromolecule grafted to a polypeptide targeting pl-CSA, the hydrophobic end of the amphiphilic macromolecule interspersed in the hydrophobic polymer layer, The hydrophilic end of the amphiphilic macromolecule is linked to the polypeptide via an amide bond, the polypeptide being exposed outside of the hydrophobic polymer layer.
  • the targeted nano delivery system is a spherical structure having a diameter of 80-150 nm.
  • the particle size is measured using a transmission electron microscope.
  • the nano-scale spheroidal delivery system helps to reduce renal excretion clearance, reticuloendothelial system absorption and phagocytic cell recognition; secondly, it can smoothly reach the target tissue through the capillary endothelial cell gap.
  • the targeted delivery system further comprises a target delivery material, the target delivery material being encapsulated by the hydrophobic polymer layer.
  • the target delivery constitutes the core of the targeted delivery system. This can effectively prevent the loaded target delivery material from aggregating or leaking before reaching the target tissue, and ensuring the stability of the loaded delivery material.
  • the target delivery object comprises at least one of a contrast agent, a fluorescence tracer, a photothermal conversion reagent, a pregnancy drug, and an antitumor drug.
  • the target delivery contains gaseous components, the targeted delivery system may be referred to as "pl-CSA targeted nanobubbles.”
  • the mass ratio of the hydrophobic polymer to the target delivery material is 1: (0.1-0.8), preferably 1: (0.1-0.5).
  • the target delivery is a mixture of at least one of a contrast agent, a fluorescence tracer, and a photothermal conversion reagent and an antitumor drug.
  • the targeted nano-delivery system can be used to diagnose, treat certain cancers or tumors associated with inappropriate expression of placenta-like chondroitin sulfate.
  • the mass ratio of the anti-tumor drug to other target delivery materials other than the anti-tumor drug is 1: (- 4), preferably 1: (0.2-3).
  • the mass ratio of the antitumor drug to the contrast agent is 1: (0.2-1), more preferably 1:1.
  • the target delivery is a mixture of at least one of a contrast agent, a fluorescence tracer, and a photothermal conversion reagent with a gestational drug.
  • the targeted nano-delivery system can be used for diagnosis, treatment and placental-like chondroitin sulfate. When expressing a related pregnancy disorder.
  • the mass ratio of the pregnancy drug to other target delivery products other than the pregnancy drug is 1: (0.1-4)
  • the mass ratio of the pregnancy drug to other target delivery products other than the pregnancy drug is 1: (0.1-4)
  • it can be 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1 or 1:2.
  • the mass ratio of the pregnancy drug to the contrast agent is 1: (1-4), more preferably 1:1.
  • the hydrophobic polymer layer is composed of a hydrophobic polymer.
  • the mass ratio of the hydrophobic polymer to the amphiphilic macromolecule is 1: (0.01-0.04).
  • the amphiphilic macromolecule can be evenly interspersed into the hydrophobic polymer layer at an appropriate density, so that the targeted nano-delivery system can be grafted with a suitable concentration of the polypeptide.
  • the degree of tightness of the hydrophobic polymer layer to the target delivery is also possible to influence the degree of tightness of the hydrophobic polymer layer to the target delivery.
  • the structure of the targeted nano-delivery system is relatively stable, the morphology is relatively regular, and the dispersibility is good.
  • the targeted nano-delivery system is not easily diluted, dissolved and disintegrated by human body fluid, and is beneficial to the targeted nano-delivery system.
  • tissue expressing placenta-like chondroitin sulfate A such as cancer cells, placental trophoblast cells, etc.
  • the mass ratio of the hydrophobic polymer to the viscous molecule is 1: (0.2-0.8).
  • the viscous molecule is selected from at least one of polyvinyl alcohol (PVA), glucose, hyaluronic acid, and gelatin.
  • the viscous molecule has certain adhesion, and is mainly used for improving the compactness and sealing property of the hydrophobic polymer layer, and avoiding the target delivery of the hydrophobic polymer layer wrapped in the later stage in the freeze-drying process.
  • the leak is revealed and it has a certain protective effect.
  • the viscous molecules can be degraded, such that the denseness of the hydrophobic polymer layer is reduced, facilitating the release of the target delivery material.
  • the amphiphilic macromolecule has a hydrophobic end and a hydrophilic end attached to the lipid end.
  • the hydrophobic end of the amphiphilic macromolecule can facilitate insertion of the amphiphilic macromolecule into the hydrophobic polymer layer, and the polypeptide is grafted and extended with the hydrophobic end of the amphiphilic macromolecule Outside of the targeted nano delivery system. More specifically, the polypeptide is exposed outside of the hydrophobic polymer layer, as well as to the exterior of the viscous molecule.
  • the mass ratio of the amphiphilic macromolecule to the polypeptide is 1: (1-5). At this mass ratio, the grafting ratio of the polypeptide to the amphiphilic macromolecule is higher. Preferably, the mass ratio of the amphiphilic macromolecule to the polypeptide is 1: (1-4).
  • a targeted nano delivery system provided in a second embodiment of the second aspect of the invention has a hydrophilic outer shell that targets pl-CSA, which can have a tissue that is inappropriately expressed for pl-CSA Good targeting.
  • the single layer lipid molecule is selected from the group consisting of lecithin and cephalin At least one of (phosphatidylethanolamine) selected from one or more of the group consisting of soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin, and phosphatidylcholine. Further preferably, the single layer lipid molecule has a hydrophobic portion facing the hydrophobic core and a hydrophilic portion facing the outside of the nanobubble.
  • the amphiphilic macromolecule is a polyethylene glycol-derivatized phospholipid obtained by linking polyethylene glycol and its derivatives via a covalent bond and a phospholipid.
  • the hydrophobic end of the amphiphilic macromolecule is the phospholipid substance, the hydrophilic end is a carboxyl group or an amino group modified polyethylene glycol, or a polyethylene glycol derivative having other reactive functional groups.
  • Its hydrophilic end-polyglycol (PEG) can effectively block the recognition of the nanobubbles by the immune system, significantly prolong the circulation time of the nanobubbles in the body, and then enriched by the enhanced permeation retention effect (EPR effect). In the placental tissue, passive targeting is finally achieved. Further based on active targeting of the above polypeptides and passive targeting by PEG, the targeted delivery system has a strong affinity for target tissues expressing pl-CSA.
  • the molecular weight of the polyethylene glycol is preferably 200 to 20000.
  • the molecular weight of the polyethylene glycol molecule may be 200, 500, 1000, 2000, 5000, 7000, 10000, 15000 or 20000.
  • the phospholipids may be synthetic or naturally occurring phospholipids, which may be, but are not limited to, distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidylglycerol (DSPG) or cholesterol.
  • the amphiphilic macromolecule is distearoylphosphatidylethanolamine-polyethylene glycol-carboxylic acid copolymerization (DSPE-PEG-COOH, also known as phospholipid-PEG-carboxyl), distearoyl phospholipid -PE - polyethylene glycol - amino copolymer (DSPE-PEG-NH 2, also known as phospholipid -PEG- amino), or distearoyl phosphatidyl ethanolamine - polyethylene glycol - maleimide.
  • DSPE-PEG-COOH also known as phospholipid-PEG-carboxyl
  • DSPE-PEG-NH 2 distearoyl phospholipid -PE - polyethylene glycol - amino copolymer
  • distearoyl phosphatidyl ethanolamine - polyethylene glycol - maleimide distearoylphosphatidylethanolamine-polyethylene glycol-carboxylic acid copolymerization
  • the hydrophobic polymer is selected from one or more of polylactic acid-glycolic acid copolymer (also known as polyglycolide lactide, abbreviated as PLGA), polylactic acid and polycaprolactone, but Not limited to this.
  • polylactic acid-glycolic acid copolymer also known as polyglycolide lactide, abbreviated as PLGA
  • polylactic acid and polycaprolactone but Not limited to this.
  • the hydrophobic polymer is a polylactic acid-glycolic acid copolymer (abbreviated as PLGA), and the PLGA has a molecular weight of 7,000 to 17,000. Wherein, the copolymerization ratio of the monomeric lactic acid to the glycolic acid is 50:50.
  • PLGA polylactic acid-glycolic acid copolymer
  • the monolayer lipid molecule is selected from at least one of lecithin and cephalin (phosphatidylethanolamine) selected from the group consisting of soy lecithin, hydrogenated soy lecithin, egg yolk lecithin and phosphatidylcholine One or more of the bases.
  • the single layer lipid molecule has a hydrophobic portion facing the hydrophobic core and a hydrophilic portion facing the exterior of the targeted delivery system.
  • the targeted delivery system further comprises: a serum albumin layer and a saccharide molecule, the saccharide molecule being adhered to the serum albumin layer, the serum The polypeptide targeting pl-CSA is grafted onto the albumin layer, and the polypeptide is grafted to serum albumin in the serum albumin layer by specific binding of biotin-avidin.
  • the targeted delivery system is spherical and has a diameter on the order of microns.
  • the targeted delivery system has a diameter of from 2 to 10 [mu]m.
  • a micron-scale spherical targeted delivery system that can be targeted to the surface of a particular tissue by the outermost polypeptide.
  • the targeted delivery system further comprises a target delivery substance, the target delivery substance being encapsulated by the serum albumin layer; the target delivery substance constitutes an inner core of the delivery system, and the target delivery substance may be embedded with a saccharide
  • the serum albumin layer of the molecule is tightly packed, which can effectively prevent the target delivery substance from aggregating or leaking before reaching the target tissue, and ensuring the stability of the target delivery of the load.
  • the target delivery object comprises at least one of a contrast agent, a fluorescence tracer, a photothermal conversion reagent, a pregnancy drug, and an antitumor drug.
  • the targeted delivery system can be used to diagnose, treat certain pregnancy diseases, cancers or tumors associated with inappropriate expression of placenta-like chondroitin sulfate.
  • the targeted delivery system when the target delivery contains a gaseous component, the targeted delivery system may be referred to as "pl-CSA targeting microbubbles".
  • the mass ratio of the serum albumin to the target delivery substance is 1: (0.1-0.8).
  • the pregnancy drug and the anti-tumor drug are not present at the same time. Further preferably, the pregnancy drug and the anti-tumor drug are not present at the same time.
  • the target delivery substance is one of the pregnancy drug and the anti-tumor drug, and a mixture of at least one of a contrast agent, a fluorescence tracer, and a photothermal conversion reagent,
  • the mass ratio of the antitumor drug to other target delivery materials other than the antitumor drug is 1: (0.1-4), preferably 1: (0.2- 3).
  • the mass ratio of the antitumor drug to the contrast agent is 1: (0.2-1), more preferably 1:1.
  • serum albumin is at least one of human serum albumin, bovine serum albumin, porcine serum albumin, and egg albumin, but is not limited thereto.
  • the saccharide molecule is at least one of glucose, fructose, sucrose, and maltose, but is not limited thereto.
  • the saccharide molecule has certain adhesion, and is mainly used for improving the compactness and sealing property of the serum albumin layer, making it difficult to be disintegrated, and avoiding early leakage of the target delivery substance wrapped by the serum albumin layer in the later stage. Come out and play a protective role.
  • the carbohydrate molecule can be degraded again, so that the density of the serum albumin layer is lowered, which is convenient for the target. The delivery is released near the target tissue.
  • the saccharide molecule is chimeric in the serum albumin layer.
  • the serum albumin and sugar The mass ratio of the molecules is 1: (2-8).
  • the polypeptide in the targeted delivery system, is labeled with avidin, and the outer surface of the serum albumin layer is adsorbed with biotin, so that the polypeptide passes between avidin and biotin.
  • the specific binding force is grafted onto the serum albumin layer.
  • the avidin comprises at least one of avidin and streptavidin.
  • the mass ratio of serum albumin to polypeptide is 1: (1-5).
  • it can be 1:1, 1:2, 1:3, 1:4 or 1:5.
  • the mass ratio of the biotin to the serum albumin is 1: (100-1000), such as 1:200, 1:400, 1:500, 1:600 or 1:800.
  • a suitable concentration of the polypeptide can be grafted into the serum albumin layer in the targeted delivery system to uniformly distribute the polypeptide outside the serum albumin layer for the targeted delivery system. It is possible to provide more uniform target sites and to avoid waste of raw materials.
  • serum albumin can self-assemble into a serum albumin layer, which can be used to wrap a target delivery substance, and the sugar molecule can be adhered. Attached to the serum albumin layer to provide a density, the serum albumin layer is further grafted with a polypeptide targeting pl-CSA, and the polypeptide is specifically reacted with the serum by biotin-avidin.
  • the serum albumin phase grafting in the albumin layer provides the targeted delivery system with a specific receptor that targets pl-CSA, and thus, the targeted delivery system can be inappropriately expressed for pl-CSA Have better targeting.
  • the targeted delivery system carries the target delivery, they can be specifically supplied to the target tissue to improve the diagnostic or therapeutic effect.
  • the targeted delivery system comprises: a pharmaceutical carrier and a loaded target delivery thereof, wherein the pharmaceutical carrier is covalently linked to the polypeptide targeting pl-CSA Inorganic nanomaterials comprising graphene oxide, oxidized carbon nanotubes, carboxylated phosphoenene, carboxylated mesoporous silicon, or aminated mesoporous silicon.
  • the mass ratio of the inorganic nanomaterial to the polypeptide is 1: (5-30). At this ratio, the graft ratio of the polypeptide to the inorganic nanomaterial is higher and the amount is less.
  • the inorganic nanomaterial is at least one of oxidized multi-walled carbon nanotubes having a diameter of 50 to 500 nm and oxidized single-walled carbon nanotubes having a diameter of 1 to 100 nm.
  • the inorganic nanomaterial is carboxylated mesoporous silicon having a diameter of 80 to 150 nm, and the carboxylated mesoporous silicon has a pore diameter of 2 to 50 nm.
  • the target delivery substance is bound to the surface of the drug carrier by physical adsorption (or "non-covalent bond").
  • the target delivery object comprises at least one of a fluorescent tracer, a contrast agent, a photothermal conversion reagent, a pregnancy drug, and an antitumor drug.
  • the target delivery is a non-gaseous component.
  • the targeted delivery system can be used for diagnosis, treatment, and at this time, the targeted delivery system can be used to diagnose, treat certain pregnancy diseases, cancers, or tumors associated with inappropriate expression of pl-CSA.
  • the mass ratio of the drug carrier to the target delivery product is 1: (3-5), and at this mass ratio, the target delivery material has a higher loading rate on the drug carrier and can be stably present.
  • the target delivery is an anti-tumor drug and a fluorescent tracer.
  • the mass ratio of the antitumor drug to the fluorescent tracer is 1: (1-4), and at this ratio, the anticancer drug and the fluorescent tracer can exert the maximum diagnostic and therapeutic effects.
  • the surface of the drug carrier is covalently modified with a receptor polypeptide having a specific targeting property for pl-CSA, such that the targeted delivery system It has better targeting and enrichment for tissues that are not properly expressed by pl-CSA.
  • the target delivery material has good stability in the targeted delivery system, and the loaded target delivery substance (such as chemotherapeutic drug, fluorescent tracer, etc.) can exclusively reach the target tissue such as a tumor, and is released in the vicinity thereof to improve the utilization of the drug.
  • the targeted delivery system helps to improve the diagnostic or therapeutic effect on diseases that are inappropriately expressed with pl-CSA.
  • the targeted delivery system further comprises a small molecule drug residue, the small molecule drug residue being linked to the polypeptide via a linker group, wherein A small molecule drug residue refers to a residual portion of a small molecule drug that removes an active group that does not affect its drug activity; the small molecule drug includes a contrast agent, a fluorescent tracer, a photothermal conversion reagent, a pregnancy drug, and an antitumor drug. At least one of them.
  • the small molecule drugs here are non-gaseous ingredients.
  • the chemical bond connecting the linker group to the small molecule drug residue comprises an amide bond (-NH-CO-), or an ester bond (-O-CO-); the linker group and the The polypeptides are linked by an amide bond.
  • linker group is -NH-CO-(CH 2 ) n -CO-* or -O-CO-(CH 2 ) n -CO-*, and the * terminus of the linker group Covalently linked to the terminal amino group of the polypeptide.
  • the targeted delivery system provided by the second aspect of the invention can have better targeting to tissues that are not properly expressed by pl-CSA.
  • the above antitumor drugs and pregnancy drugs include one or more of various chemical drugs, polypeptide drugs, proteins, vaccines, and gene drugs.
  • the "chemical drug” includes but is not limited to an organic compound; the “gene drug” includes but It is not limited to cationic polymers, polypeptides, polyamino acids or transfection reagents that encapsulate, bind or blend nucleic acid fragments.
  • polypeptide drugs, proteins, vaccines and gene drugs can be referred to as "biopharmaceuticals”.
  • pregnancy drugs are for pregnancy diseases.
  • Pregnancy diseases refer to pregnancy-related diseases that occur during pregnancy, such as intrauterine growth retardation, gestational diabetes, and premature labor.
  • pregnancy drugs include treatment of gestational diabetes, treatment of pregnancy syndrome, treatment of intrauterine growth retardation, premature delivery, treatment of pre-eclampsia (also known as "pre-eclampsia”) and prevention of premature rupture of membranes of the above-mentioned various types of chemicals, organisms drug.
  • the pregnancy drug is selected from the group consisting of sulphate, progesterone, misoprostol, indomethacin, relaxing peptide, digoxigenin antibody, digitalis antibody, growth hormone-like factor 2, insulin growth factor 2
  • the ELABELA polypeptides are not limited thereto.
  • premature gestational drugs also known as "abortion drugs” may be selected from misoprostol, mifepristone, prostaglandin, thioprostone, tamoxifen, letrozole and methotrexate. One or more of them.
  • the anti-tumor chemical drugs may include doxorubicin, epirubicin, paclitaxel, norvinblastine, etoposide, cisplatin, methotrexate, curcumin, 5-fluorouracil, and bacterium red And one or more of them in a pharmaceutically acceptable salt, but are not limited thereto.
  • doxorubicin hydrochloride is a pharmaceutically acceptable salt of doxorubicin and also has certain antitumor activity.
  • the anti-tumor drug is doxorubicin, doxorubicin hydrochloride, epirubicin, epirubicin hydrochloride or paclitaxel.
  • the anti-tumor polypeptide drug may be selected from the group consisting of Triptorium, ES-2 polypeptide, scorpion venom polypeptide, melittin, leuprolide, buserelin, soybean peptide, pea peptide, At least one of egg white peptide, polymyxin, lactobacillus, nisin, bacitracin, actinomycin, and bleomycin, but is not limited thereto.
  • fluorescent tracer indigo green, Evans blue, isosulfan blue, patent blue, methylene blue, coumarin 6, IR780 iodide (11-chloro-1,1'-di-n-propyl group) One or more of -3,3,3',3'-tetramethyl-10,12-trimethylenesulfonium tricarbon cyanine iodide) and DiR iodide, but is not limited thereto.
  • the contrast agent comprises at least one of an X-ray contrast agent, a magnetic resonance imaging contrast agent, and an ultrasound contrast agent.
  • Ultrasound contrast agents are preferably used to improve the convenience of contrast imaging.
  • an ultrasound contrast agent it may include at least one of a biological inert gas, a liquid fluorocarbon, and a thermosensitive gas generating agent.
  • the biological inert gas is selected from the group consisting of nitrogen, sulfur hexafluoride, perfluoropropane (C 3 F 8 ), perfluorobutane, etc.
  • the liquid fluorocarbon may be selected from perfluorohexane (ie, fourteen Fluorohexane, C 6 F 14 ), perfluorooctyl ammonium bromide (PFOB), perfluorohexane (PFP), perfluorodecalin (PFD), etc.
  • the thermosensitive gas generating agent may be selected from calcium carbonate (CaHCO 3 ), ammonium hydrogencarbonate (NH 4 HCO 3 ), etc., but is not limited thereto.
  • the X-ray contrast agent may, for example, be one or more of iodobenzene hexaol, iopromide, iodine, iodophenyl ester and barium sulfate, but is not limited thereto.
  • iodobenzene hexaol iopromide
  • iodine iodophenyl ester
  • barium sulfate but is not limited thereto.
  • photothermal conversion reagent examples include indocyanine green, carbon nanotubes, gold nanoparticles, gold nanotubes, and the like.
  • the tissue that improperly expresses pl-CSA is more targeted, and the degree of enrichment in the target tissue is high; when the target is loaded When the product is delivered, the stability of the load can be improved, the circulation time in the body can be prolonged, and the detection, treatment, and the like of the disease associated with inappropriate expression of pl-CSA can be used.
  • the invention also provides a method of making a targeted delivery system according to several embodiments of the second aspect of the invention.
  • the preparation method is as follows:
  • the second mixed solution further contains a target delivery substance
  • the target delivery substance includes at least one of a contrast agent, a fluorescence tracer, a photothermal conversion reagent, a pregnancy drug, and an antitumor drug.
  • the second mixed solution is prepared as follows: the hydrophobic polymer solution is added dropwise to the first After the solution is mixed, a target delivery of the gaseous component is introduced into the first mixed solution, and after shaking, a second mixed solution is obtained.
  • the oscillation can contribute to the dissolution of the gaseous target delivery, and it can facilitate the encapsulation of more gaseous target delivery in the hydrophobic core. Further, the time of the oscillation is 60 s-2 min.
  • a mixture of the hydrophobic polymer solution and the first mixed solution ie, containing a single layer of lipid molecules, an amphiphilic macromolecular compound, and hydrophobic multimerization
  • the mixture of substances is transferred to a sealed bottle (for example, a vial), and the air in the vial is replaced with a biologically inert gas, which is oscillated.
  • the hydrophobic target delivery is added to the hydrophobic polymer solution. That is, the target delivery material of the hydrophobic non-gaseous component is also dissolved in the organic solvent, and at this time, the hydrophobic polymer solution contains a hydrophobic target delivery product.
  • the hydrophilic target delivery is added to the first mixed solution. That is, the hydrophilic target delivery material is also dissolved in the first solvent A. At this time, the first mixed solution contains a hydrophilic target delivery product).
  • the non-gaseous components may include target delivery materials in liquid and/or solid form.
  • the above hydrophobic component means a substance which is hardly compatible with water;
  • the hydrophilic component herein means a liquid and/or solid component which is non-hydrophobic (including amphiphilic).
  • the preparation process of the second mixed solution is as follows :
  • a single layer of a lipid molecule, an amphiphilic macromolecular compound, and a hydrophilic non-gaseous target delivery substance are dissolved in the first solvent A to obtain a first mixed solution; and the hydrophobic polymer solution is further added to In the first mixed solution, a target delivery substance of a gaseous component is then introduced into the first mixed solution, and after shaking, a drug-loaded suspension solution is obtained.
  • the preparation process of the second mixed solution is as follows:
  • a hydrophobic polymer solution Dissolving the hydrophobic polymer and the hydrophobic target delivery substance in an organic solvent to obtain a hydrophobic polymer solution; dissolving the monolayer lipid molecule, the amphiphilic macromolecular compound and the hydrophilic target delivery substance In the first solvent A, a first mixed solution is obtained; the hydrophobic polymer solution is further added to the first mixed solution, and after mixing, a second mixed solution is obtained.
  • the organic solvent comprises one or more of acetonitrile, acetone, diethyl ether, chloroform, dichloromethane and n-hexane.
  • the organic solvent is preferably a volatile solvent capable of dissolving a hydrophobic polymer.
  • the first solvent A comprises at least one hydrophilic solvent or a mixed solvent of water and at least one hydrophilic solvent.
  • the hydrophilic solvent is selected from the group consisting of ethanol, methanol, 1-octanol, acetonitrile, acetone, dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), but is not limited thereto.
  • the first solvent A is required to dissolve both the amphiphilic macromolecular compound, the monolayer lipid molecule, and the non-hydrophobic target delivery material.
  • the first solvent A is a mixed solvent of water and at least one hydrophilic solvent.
  • the first solvent A contains water and can reduce the solubility of the hydrophobic polymer when it is mixed with the organic solvent solution of the hydrophobic polymer in the later stage. It can facilitate post-ultrasound and emulsification into balls.
  • the first solvent A may be an aqueous solution of various concentrations of ethanol, various concentrations of aqueous methanol. Further preferably, in the first solvent A, the volume fraction of water is 3-8%. In an embodiment of the invention, the first solvent A is an aqueous ethanol solution having a volume fraction of 4% or an aqueous methanol solution having a volume fraction of 4%.
  • the concentration of the single layer lipid molecule is 10-300 ⁇ g/mL, and the concentration of the amphiphilic macromolecule compound is 30-600 ⁇ g/mL. More preferably, it is 100-500 ⁇ g / mL.
  • the concentration of the hydrophobic polymer solution is 1-4 mg/ml.
  • step (3) the volume ratio of the hydrophobic polymer solution to the first mixed solution is 1:3.
  • the hydrophobic polymer solution is mixed with the first mixed solution in a dropwise addition manner.
  • the hydrophobic polymer solution has a dropping rate of 0.2 to 0.5 mL/min. In this way, the hydrophobic polymer can be fully complexed with the target delivery material and wrapped into the outer casing, and the ultrasound can be combined to form a drug-loaded targeted delivery system with more stable structure and higher loading efficiency.
  • the ultrasonication is performed using an ultrasonic cell disrupter at a frequency of 80-160 W at a frequency of 20 kHz.
  • the second mixed solution is gently stirred at 40-80 °C.
  • gentle agitation provides suitable solvent evaporation conditions so that the resulting targeted delivery system precursor has better dispersibility and a more uniform particle size.
  • the hydrophobic polymer, the target delivery substance, the monolayer lipid molecule, and the amphiphilic macromolecular compound form the target delivery system precursor by a self-assembly process (ie, non-targeting nanobubbles) ), no chemical reaction is required, and the preparation process is environmentally friendly and non-toxic, and the method is simple and easy to operate.
  • a self-assembly process ie, non-targeting nanobubbles
  • the centrifugation is carried out 2 to 5 times in an ultrafiltration centrifuge tube having a molecular weight cut off of 5 to 10 kDa.
  • water was used for washing after each centrifugation.
  • the centrifugation is performed at a centrifugal speed of 3000-5000 rpm for 3-6 min each time.
  • the amide reaction can be carried out at room temperature or at 3 to 10 °C.
  • the amide reaction is carried out for a period of from 15 to 24 hours. It is preferably 15-20 h.
  • the target nano delivery system precursor is added to the second solvent A during the amide reaction, and the catalyst and the dehydrating agent are added. After activation for 0.5-3 h, the polypeptide targeting the placental chondroitin sulfate is further added, and the reaction is stirred for 15-24 hours (preferably 18-24 hours) to obtain a reaction solution.
  • the amphipathic macro part is added first upon activation. a sub-compound, a catalyst, a dehydrating agent; then, after the target nanoparticle precursor is added, the reaction is carried out for 15-24 hours with stirring to obtain a reaction liquid.
  • the method further comprises: separating and purifying the reaction liquid to obtain a pl-CSA targeted nano delivery system.
  • the separation and purification are carried out by ultrafiltration centrifugation using an ultrafiltration centrifuge tube having a molecular weight cut off of 5-10 kDa, and the supernatant obtained after centrifugation is collected.
  • the ultrafiltration is performed 2-5 times, except for the last ultrafiltration centrifugation, followed by washing with water or PBS after each centrifugation.
  • the ultrafiltration centrifugation is performed at a centrifugal speed of 3000-5000 rpm for 3-6 min each time.
  • the second solvent A may be water or other hydrophilic solvent.
  • the second solvent A comprises water, 2-(N-morpholine)ethanesulfonic acid buffer (referred to as "MES buffer solution”) having a pH of 5.5 to 6.7, and phosphoric acid having a pH of 7.0 to 7.9. Salt (PBS) buffer, etc., but is not limited thereto.
  • MES buffer solution 2-(N-morpholine)ethanesulfonic acid buffer
  • PBS Salt
  • the method of the amidation reaction is well known to those skilled in the art.
  • the catalyst which may also be referred to as an activator, is often used in combination with a condensing agent for the amidation reaction.
  • the condensing agent comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (abbreviated as EDC).
  • EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • the catalyst includes any one of N-hydroxysuccinimide (NHS) and N-hydroxysulfosuccinimide sodium salt (Sufo-NHS).
  • the mass ratio of the condensing agent, the catalyst to the amphiphilic macromolecular compound is (0.2-0.4): (0.05-0.3):1. More preferably, when the amphiphilic macromolecular compound is DSPE-PEG-COOH, the mass ratio of the EDC, NHS, DSPE-PEG-COOH is 1:0.4:5.
  • a precursor of a targeted delivery system that does not have an active targeting function is prepared, and finally a polypeptide that targets placenta-like chondroitin sulfate A is grafted, compared to the polypeptide first.
  • the preparation method provided by the invention can greatly reduce the ultrasound and the like. The effect of energy on the activity of the polypeptide.
  • the polypeptide may also be grafted onto the amphiphilic macromolecular compound, and then the hydrophobic polymer, the target delivery substance, and the polypeptide grafted parent.
  • the macromolecular compound, the monolayer lipid molecule is prepared according to the procedures of steps (1)-(3).
  • the preparation method of the targeted delivery system according to the first embodiment of the second aspect of the present invention provided above is simple and easy to operate, and the particle size of the obtained targeted delivery system is controllable and uniform.
  • the preparation method is as follows:
  • the targeted delivery system precursor subjecting the targeted delivery system precursor to the polypeptide, the catalyst, and the dehydrating agent targeting the placenta-like chondroitin sulfate A in an amide reaction in the second solvent A, so that the polypeptide is grafted to the two On the paternally macromolecule, the targeted delivery system targeting the placenta-like chondroitin sulfate A is obtained.
  • the targeted nano delivery system further comprises a target delivery substance, the target delivery substance being encapsulated by the hydrophobic polymer layer, the target delivery substance comprising a contrast agent, a fluorescent trace agent, a pregnancy drug and an anti-antibody At least one of tumor drugs.
  • a target delivery of the gaseous component is introduced into the second mixed solution A, and then oscillated; when the target delivery contains a hydrophobic non-gaseous component Adding the hydrophobic target delivery to the hydrophobic polymer solution; when the target delivery contains a hydrophilic non-gaseous component, the hydrophilic target delivery It is added to the first mixed solution A.
  • several such addition sequences described above are advantageous for increasing the loading rate of the hydrophobic polymer to various morphological, pro-/hydrophobic target delivery materials.
  • the concentration of the hydrophobic polymer solution is 25-75 mg/mL.
  • the vibration may be carried out in a constant temperature oscillator at a temperature of 20 to 30 °C. More preferably, it is 20-25 °C.
  • the mass fraction of the emulsifier in the aqueous emulsifier is 1-3%, and the emulsifier comprises sodium cholate or polyether F68 (ie, propylene glycol block polyether).
  • the ultrasonic treatment has a power of 200-400 W, an operating voltage of 120 V, and a frequency of 20-30 kHz.
  • the evaporation is carried out in a constant temperature evaporator, and the evaporation time is 2-5 h.
  • the purpose of evaporation is to remove volatile organic solvents in the pre-emulsion, especially non-hydrophilic solvents used to dissolve the hydrophobic polymer.
  • the temperature of the evaporation may be selected depending on the boiling point of the solvent contained in the system. Alternatively, the evaporation temperature is 20-80 ° C, preferably 35-62 ° C.
  • the ultrafiltration centrifuge tube has a molecular weight cut-off of 5-10 kDa; and the ultrafiltration centrifuge has a centrifugal speed of 3000-5000 rpm, each centrifugation for 3-6 min.
  • the number of times of ultrafiltration centrifugation is 3-5 times.
  • each ultrafiltration was washed with water to remove the emulsifier.
  • the mass fraction of the viscous molecules in the aqueous solution of the viscous molecule is 1% to 3%, and the viscous molecules include PVA, glucose, fructose, sucrose, maltose, hyaluronic acid and gelatin. At least one of them.
  • the amide reaction is carried out at 3 to 10 °C.
  • the amide reaction conditions e.g., catalyst, dehydrating agent, second solvent A, etc.
  • the amide reaction conditions can be referred to the above description in the preparation method of the targeted delivery system described in the second embodiment of the second aspect.
  • the selection of the first solvent A reference may also be made to the preparation method described above.
  • the preparation method is as follows:
  • the targeted delivery system further comprises a target delivery; the target delivery comprises at least one of a contrast agent, a fluorescence tracer and an anti-tumor drug.
  • the first mixed solution B contains a target delivery substance of a hydrophilic component. That is, the serum albumin and the saccharide molecule are dissolved in the first solvent B together with the target delivery substance of the hydrophilic component to obtain the first mixed solution B.
  • the target delivery material contains a hydrophobic component
  • the target delivery material of the hydrophobic component is added to the first mixed solution B.
  • the target delivery material contains a gaseous component
  • a gaseous state is introduced into the first mixed solution B.
  • the target delivery of the fraction is then oscillated for 2-5 min.
  • the oscillation is performed in a constant temperature oscillator, and the temperature at the time of the oscillation is 20-30 °C. More preferably, it is 20-25 °C.
  • the first solvent B comprises water, 2-(N-morpholine) ethanesulfonic acid buffer (referred to as "MES buffer solution”) or pH value of pH 5.5-6.7.
  • MES buffer solution 2-(N-morpholine) ethanesulfonic acid buffer
  • PBS 7.0 to 7.9 phosphate buffer
  • physiological saline or water and at least one hydrophilic solvent (for example, methanol, ethanol, glycerol, 1-octanol, etc.), but is not limited thereto. Therefore, as long as the carbohydrate molecule and the serum ubiquitin can be dissolved at the same time.
  • hydrophilic solvent for example, methanol, ethanol, glycerol, 1-octanol, etc.
  • the mass fraction of serum albumin in the first mixed solution B is 10-40%, and the mass fraction of the saccharide molecule is 10-80%.
  • the ultrasonic treatment has a power of 200-400 W; and the frequency is 20-30 kHz.
  • the power of the sonication should not be too large to avoid a structural integrity of the serum albumin layer in the resulting targeted delivery system.
  • the frequency of the ultrasonic treatment is 20 kHz, the operating voltage is 120 V, and the power is 200-400 W.
  • the temperature at the time of standing at a low temperature is 4 to 10 °C.
  • the centrifugation speed of the centrifugation treatment is 2000-5000 rpm, and the centrifugation time is 2-6 min.
  • the centrifugal speed is further preferably from 2,500 to 3,500 rpm.
  • the centrifugation of this step is carried out in a conventional centrifuge tube rather than in an ultrafiltration tube.
  • the isotonic solution is a 0.9% NaCl solution, a MES buffer solution having a pH of 5.5 to 6.7, or a PBS buffer having a pH of 7.0 to 7.9.
  • the isotonic solution may be the same as or different from the first solvent.
  • the avidin comprises avidin and streptavidin.
  • step (3) the incubation is carried out at room temperature at a temperature of 25-37 °C.
  • the separation and purification are carried out by ultrafiltration centrifugation in an ultrafiltration centrifuge tube having a molecular weight cut off of 5-10 kDa, and washed with water or PBS, and the supernatant after centrifugation is collected to obtain the target. Delivery system. Further, the ultrafiltration centrifugation is carried out 2-5 times.
  • the centrifugal speed of the ultrafiltration centrifugation is 2000-5000 rpm, and the centrifugation time is 2-6 min.
  • the ultrafiltration centrifugal speed is further preferably from 2,500 to 3,500 rpm.
  • the preparation method is as follows:
  • an inorganic nanomaterial comprising graphene oxide, oxidized carbon nanotubes, carboxylated phosphoenene, carboxylated mesoporous silicon, or aminated mesoporous silicon;
  • the inorganic nanomaterial is amidated in the first solvent C with the polypeptide, the catalyst and the dehydrating agent targeting the placenta-like chondroitin sulfate A for 15-20 h to obtain a reaction liquid;
  • the reaction solution is centrifuged at 8000-10000 rpm, the supernatant is discarded, and the precipitate is collected to obtain a drug carrier, that is, an inorganic nanomaterial covalently linked to the polypeptide;
  • the first solvent C may be selected from the same range as the second solvent A, and includes 2-(N-morpholine) ethanesulfonic acid buffer (water) and a pH value of 5.5 to 6.7. It is a "MES buffer solution” or a phosphate buffer solution (PBS) having a pH of 7.0 to 7.9, but is not limited thereto.
  • MES buffer solution or a phosphate buffer solution (PBS) having a pH of 7.0 to 7.9, but is not limited thereto.
  • the conditions of the amide reaction in the step (1) can be referred to the above description in the preparation method of the targeted delivery system described in the second embodiment of the second aspect.
  • the second solvent B may be selected from water or a mixed solvent of at least one hydrophilic solvent and water.
  • the hydrophilic solvent is selected from the group consisting of ethanol, methanol, 1-octanol, acetonitrile, acetone, dimethylformamide (DMF), and dimethyl sulfoxide (DMSO), but is not limited thereto.
  • the second solvent is a mixed solvent, the volume fraction of water is 15-30%.
  • the shaking and mixing time is 24-48 h.
  • the ultrasonic power is 200-400 W
  • the ultrasonic time is 2-6 min.
  • the centrifugal rotation speed of the centrifugation is 10,000-15000 rpm, and the centrifugation time is 5-10 min.
  • the graphene oxide is prepared by a modified Hummers method, specifically: adding graphite powder to a mixed acid formed by mixing a concentrated nitric acid having a mass concentration of 68% and a concentrated sulfuric acid having a mass concentration of 98% at a volume ratio of 1:6. After magnetic stirring for 30 minutes in an ice bath, potassium permanganate was slowly added at 3 to 6 ° C. After the potassium permanganate was completely added, the reaction temperature was raised to 30 to 45 ° C, and the reaction was stirred for 2 hours. Add excess hydrogen peroxide to remove potassium permanganate, and centrifuge the final reaction solution at 10,000 to 15,000 rpm for 15 to 30 minutes. The resulting precipitate is diluted with deionized water and filtered until the filtrate is neutral. The precipitate is vacuum dried at 60 ° C. After that, graphene oxide is obtained.
  • a modified Hummers method specifically: adding graphite powder to a mixed acid formed by mixing a concentrated nitric acid having a mass concentration of 68%
  • the preparation process of the oxidized carbon nanotubes is as follows: adding carbon nanotubes to a mixed acid solution of concentrated sulfuric acid and concentrated nitric acid, and continuously stirring at 80-90 ° C for 4-6 hours for oxidation; adding to the obtained mixture The ice water is allowed to stand, filtered, washed, and vacuum dried to obtain oxidized carbon nanotubes, that is, surface-carboxylated carbon nanotubes.
  • oxidized carbon nanotubes that is, surface-carboxylated carbon nanotubes.
  • the surface carboxylation of mesoporous silicon and phosphonene is similar, and will not be described herein.
  • the ultrasonic dispersion of the carbon nanotubes is performed at a power of 200 to 400 W for 5 to 10 minutes.
  • the mixed acid is formed by mixing a concentrated nitric acid having a mass concentration of 68% and 98% concentrated sulfuric acid in a volume ratio of 1: (1-6).
  • the volume ratio is preferably 1: (1-3).
  • the preparation method is as follows:
  • Targeted delivery systems in this case are also peptide drug conjugates. It is worth noting that when constructing the functionalized small molecule drug, care should be taken not to affect the drug activity of the small molecule drug. Of course, the resulting polypeptide drug conjugate will still have pharmaceutically active activity.
  • the amide reaction can be carried out by using a carboxyl group thereon and an amino group at the C-terminus of the polypeptide (ie, an amino group on cysteine).
  • the amino group thereon can be used for the amide reaction with the carboxyl group at the N-terminus of the polypeptide.
  • the linker when preparing a functionalized small molecule drug having a carboxyl group, the linker may be selected from an alkyl hydrocarbon dianhydride (the molecular formula may be represented by C n+2 H 2n O 3 , and n is an integer greater than 1).
  • An alkyl hydrocarbon dianhydride having 4 to 8 carbon atoms is preferred.
  • the obtained polypeptide drug conjugate can be expressed as: a small molecule drug residue - A - polypeptide residue; wherein A is a linker group, and A can be -NH-CO-(CH 2 ) n -CO-* or -O -CO-(CH 2 ) n -CO-*, wherein the * terminus of A is linked to the polypeptide residue.
  • the polypeptide residue at this time is the portion of the polypeptide after the terminal amino group is removed.
  • the small molecule drug residue refers to a residual portion of the small molecule drug after removing the active group that does not affect its drug activity.
  • the active group For example, for doxorubicin and its pharmaceutically acceptable salts, -NH 2 or -NH 2 .HCl is removed.
  • the method of the amide reaction is well known to those skilled in the art, and it is usually necessary to add a condensing agent, a catalyst (also referred to as an activator).
  • a condensing agent also referred to as an activator
  • the reaction course of the amide reaction is as follows: the functionalized small molecule drug is dissolved with a polypeptide, a solvent, a condensing agent, and a catalyst that target placenta-like chondroitin sulfate A, and the reaction is stirred. From 1 to 6 h, a polypeptide drug conjugate targeting pl-CSA was obtained.
  • the condensing agent comprises O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), O-(N-succinimide group) - bis(dimethylamino)carbonate tetrafluoroborate (TSTU), 2-(7-oxobenzotriazole)-N,N,N',N'-tetramethyluron hexafluorophosphate At least one of (HATU) and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), but is not limited thereto.
  • TBTU O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate
  • TSTU O-(N-succinimide group) - bis(dimethylamino)carbonate tetrafluoroborate
  • the catalyst includes any one of N,N-diisopropylethylamine (DIEA), N-methylmorpholine, and triethylamine (TEA), but is not limited thereto.
  • the solvent includes at least one of N,N-dimethylformamide (DMF), acetone, and tetrahydrofuran (THF), but is not limited thereto.
  • the functionalized small molecule drug when it has a carboxyl group, it may be mixed with a condensing agent and a solvent, and then the catalyst is added dropwise, stirred for 0.5-2 h, and then the target placenta-like chondroitin sulfate A is added. The polypeptide was stirred for 1-4 h, the reaction was stopped, and the reaction solution was harvested.
  • the polypeptide targeting the placenta-like chondroitin sulfate A may be mixed with a condensing agent and a solvent, and then the catalyst is added dropwise and stirred for 0.5-2 h. Then, a functionalized small molecule drug with an amino group was added, stirring was continued for 1-4 h, the reaction was terminated, and the reaction solution was harvested. Further, after the amide reaction was carried out to obtain a reaction liquid, the reaction liquid was purified by high performance liquid chromatography.
  • the preparation method of the polypeptide drug conjugate provided by the fifth embodiment of the second aspect of the present invention is simple and easy to operate.
  • the prepared polypeptide drug conjugate is more targeted to tissues that do not properly express pl-CSA, and has a high degree of enrichment in the target tissue.
  • the preparation methods of several pl-CSA targeted nano delivery systems provided by the present invention are simple and easy to operate.
  • the present invention provides the use of a targeted delivery system according to the second aspect of the invention for the manufacture of a medicament for the prevention, diagnosis or treatment of a disease associated with inappropriate expression of placenta-like chondroitin sulfate A.
  • the diseases associated with expression or inappropriate expression of pl-CSA include pregnancy diseases, tumor diseases, arthritis, joint diseases, multiple sclerosis, pathological conditions caused by nerve damage (for example, healing after nerve injury), A condition of cartilage and scar tissue (such as rheumatism, cartilage repair or wound healing), psoriasis, etc., but is not limited thereto. It should be noted that when it is desired to prevent or treat a certain disease associated with expression or inappropriate expression of pl-CSA, the target delivery product in the above targeted delivery system can be replaced accordingly as needed.
  • pregnancy diseases include pre-eclampsia (also referred to as “pre-eclampsia”), intrauterine growth retardation, premature rupture of membranes, premature delivery, gestational diabetes, pregnancy syndrome, and the like, but are not limited thereto.
  • the tumor diseases include placental villus cancer, breast cancer, pancreatic cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, lung cancer, colon cancer, prostate cancer, cervical cancer, testicular cancer, basal cell skin cancer, and transparent
  • One of renal cell carcinoma, head and neck squamous cell carcinoma, cutaneous squamous cell carcinoma, vulvar squamous cell carcinoma, vulvar basal cell carcinoma, neuroendocrine carcinoma, sarcoma, hematopoietic cancer, and neuroepithelial tissue A variety, but not limited to this.
  • the sarcoma includes, but is not limited to, fibrosarcoma, dedifferentiated cartilage and liposarcoma, leiomyosarcoma, liposarcoma, mucinous liposarcoma, uterine leiomyosarcoma, osteosarcoma, Ewing sarcoma and rhabdomyosarcoma, synovial sarcoma, isolated Fibroids; including, but not limited to, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B cells, T cells, and large granular lymphoma; Tumors of neuroepithelial tissue, including but not limited to astrocytomas (polymorphic yellow astrocytoma, fibroblastic astrocytoma, interstitial) Astrocytoma, glioblastoma multiforme, oligodendroglioma, ependym
  • the present invention provides a pharmaceutical preparation for treating a tumor, the pharmaceutical preparation for treating a tumor comprising the polypeptide according to the first aspect of the invention or the first to fifth embodiments of the second aspect of the invention Any of the targeted delivery systems described.
  • the present invention provides a pharmaceutical preparation for treating a pregnancy disease, the pharmaceutical preparation for treating a pregnancy disease comprising the polypeptide according to the first aspect of the invention or the first to fifth embodiments of the second aspect of the invention
  • the pharmaceutical preparation for treating a pregnancy disease comprises a targeted delivery system according to the first embodiment of the second aspect of the invention, a targeted delivery system according to the second embodiment of the second aspect of the invention or The targeted delivery system of the fifth aspect of the second aspect of the invention
  • Example 1 is a schematic structural view of a targeted nano delivery system prepared in Example 1;
  • Example 2 is a transmission electron micrograph of the targeted nano delivery system prepared in Example 1;
  • Example 3 is a schematic structural view of a targeted delivery system prepared in Example 7;
  • Example 4 is a particle size distribution diagram of a targeted delivery system in Example 8.
  • Figure 5 is a Zeta potential diagram of the targeted delivery system of Example 9;
  • Example 6 is a transmission electron micrograph of the targeted delivery system of Example 10.
  • Figure 7 is a graph showing the uptake results of human placental villous cancer cells against the targeted nano-delivery system and other experimental groups prepared in Example 1 of the present invention.
  • Figure 8 is a result of ingestion of different cancer cells to the targeted nano delivery system prepared in Example 1 of the present invention.
  • Figure 9 is a graph showing the therapeutic effect of the targeted nano-delivery system prepared in Example 1 of the present invention on mouse choriocarcinoma;
  • Example 10 is a diagnostic tumor distribution map of a targeted nano delivery system prepared in Example 2 of the present invention.
  • Figure 11 is a result of ultrasonic development of a targeted abortion system prepared by the targeted nano delivery system and other experimental groups prepared in Example 6 of the present invention.
  • Figure 12 is a graph showing the effect of the targeted nano delivery system and other experimental groups prepared in Example 6 of the present invention on embryo body weight;
  • Figure 13 is an in vitro contrast imaging of the targeted delivery system prepared in Example 10;
  • Example 14 is a schematic structural view of a targeted nano delivery system prepared in Example 11;
  • Figure 15 is a particle size distribution diagram of the targeted nano delivery system of Example 12.
  • Figure 16 is a transmission electron micrograph of the targeted nano delivery system of Example 13;
  • 17 is an in vitro angiographic image of (a) a non-targeted nano-delivery system and a targeted nano-delivery system (b) of Example 11 on mouse ovarian cancer tissue;
  • Figure 18 is a schematic view showing the structure of a targeted micro-delivery system prepared in Example 14;
  • Figure 19 is a graph showing the in vitro killing effect of the targeted micro-delivery system prepared in Example 14 on breast cancer cells;
  • Figure 20 is a schematic illustration of the synthesis of the targeted delivery system of Example 16.
  • Example 21 is a transmission electron micrograph of the targeted delivery system of Example 17.
  • Figure 22 is a diagram showing the drug release of the targeted delivery system prepared in Example 16 at different pH
  • Figure 23 is an ingestion plot of A549 cells to the targeted delivery system and non-targeted delivery system of Example 16;
  • 24 is a schematic diagram showing the synthesis of a polypeptide drug conjugate targeting pl-CSA according to an embodiment of the present invention.
  • Figure 25 is a graph showing the antitumor effect of different concentrations of free drug and different concentrations of the targeted polypeptide drug conjugate prepared in Example 20.
  • the sequence of the polypeptide for targeting placenta-like chondroitin sulfate A (pl-CSA) is shown in SEQ ID NO: 1 - SEQ ID NO: 3.
  • LKPSHEKKNDDNGKKLCKAC is shown as SEQUENCE NO.
  • EDVKDINFDTKEKFLAGCLIVSFHEGKC is shown as SEQUENCE NO.
  • GKKTQELKNIRTNSELLKEWIIAAFHEGKC is shown as SEQUENCE NO.
  • the polypeptide is carried out according to a conventional polypeptide synthesis process, wherein the leftmost end of each sequence is N-terminal, the rightmost end is the C-terminus of the polypeptide, and the C-terminus or the N-terminus can be combined with the substance to be grafted (such as the above-mentioned amphiphilic Molecules, avidin, inorganic Nanomaterials, functionalized small molecule drugs) are covalently linked, depending on the nature of the material to be grafted.
  • the substance to be grafted such as the above-mentioned amphiphilic Molecules, avidin, inorganic Nanomaterials, functionalized small molecule drugs
  • the amide reaction can be carried out by using a carboxyl group thereon and an amino group at the C-terminus of the polypeptide (i.e., an amino group on cysteine C).
  • an amide reaction can be carried out by using an amino group thereon and a carboxyl group at the N-terminus of the polypeptide.
  • the first targeted delivery system targeting pl-CSA and its preparation are described below.
  • Example 1 A method of preparing a targeted delivery system comprising:
  • polylactic acid-glycolic acid copolymer (PLGA, molecular weight of 15000, monomeric lactic acid and glycolic acid copolymerization ratio of 50:50) dissolved in acetonitrile to obtain PLGA in acetonitrile solution, the concentration of 2mg / mL;
  • the sonicated solution was subjected to ultrafiltration centrifugation in an ultrafiltration centrifuge tube with a molecular weight cut off of 10 kDa, and washed with water, 4 times, wherein the centrifuge was rotated at 4000 rpm for 4 min each time, and the supernatant was collected to obtain a target delivery system precursor. ;
  • the targeted delivery system is a spherical particle comprising a hydrophobic core 1, a single layer of lipid molecular layer 2 encapsulating the hydrophobic core, and a hydrophilic outer shell 3 targeting pl-CSA, a single layer of lipid molecular layer 2
  • the composition is soybean lecithin
  • the hydrophilic outer shell 3 is composed of polypeptide 32 grafted DSPE-PEG 31, 11 is hydrophobic polymer PLGA, 12 is the target delivery material - doxorubicin, 11 is 12 winding 1 is a hydrophobic core composed of 11 and 12; in the peptide-grafted DSPE-PEG, the lipid terminal DSPE of DSPE-PEG 31 is inserted into the soybean lecithin layer 22, and the hydrophilic end PEG and the polypeptide 32 pass through The amide bond is attached and the polypeptide 32 is exposed outside of the monolayer lipid molecular layer
  • Example 2 is a transmission electron microscope (TEM) image of the targeted delivery system prepared in Example 1 of the present invention.
  • the prepared targeted delivery system is spherical particles with good dispersibility and average particle size. It is 80-100nm.
  • EN% (1-Cf/Ct) ⁇ 100%, wherein Cf is the amount of free drug and Ct is the total amount of drug, and the targeted nanoparticle is obtained.
  • EN% encapsulation efficiency of doxorubicin was 37.2 ⁇ 1.54%.
  • the connectivity of the polypeptide was determined by the BCA method to be 47.3 ⁇ 5.1%.
  • Example 2 A pl-CSA targeted delivery system and a preparation method for co-encapsulating doxorubicin and phthalocyanine green are provided, which differ from the embodiment 1 in that the first mixed solution in the step (2) contains It is 750 ⁇ g of phthalocyanine green (ICG) and 750 ⁇ g of doxorubicin.
  • ICG phthalocyanine green
  • Example 3 A targeted delivery system and method of preparation are provided that differ from Embodiment 1 in that:
  • step (1) a polycaprolactone (molecular weight: 10,000) acetone solution having a concentration of 1 mg/mL is prepared; in the step (2), the first mixed solution contains 40 ⁇ g of egg yolk lecithin and 100 ⁇ g of DSPE-PEG- NH 2 (PEG molecular weight is 3000), 250 ⁇ g of lycopene; in step (4), 0.4 mg of the polypeptide as shown in SEQUENCE NO. 1 and 20 ⁇ g of EDC and 5 ⁇ g of NHS were added.
  • Example 4 A targeted delivery system and method of preparation are provided that differ from implementation 1 in that:
  • a polylactic acid (molecular weight: 21800) dichloromethane solution having a concentration of 4 mg/mL is prepared; in the step (2), the first mixed solution contains 800 ⁇ g of cephalin and 1600 ⁇ g of DSPE-PEG-NH. 2 (the molecular weight of PEG is 3000), 3000 ⁇ g of paclitaxel; in step (4), 1.6 mg of the polypeptide shown by SEQUENCE NO. 2 and 640 ⁇ g of EDC and 80 ⁇ g of NHS are added.
  • the TEM particle size of the targeted delivery system of Example 4 was 120-130 nm; the connectivity of the polypeptide was determined by the BCA method: 52.3 ⁇ 3.2%.
  • Example 5 provides a targeted delivery system and method of preparation that differs from implementation 2 in that:
  • the first mixed solution contains 800 ⁇ g of phosphatidylcholine, 1600 ⁇ g of DSPE-PEG-COOH (having a molecular weight of PEG of 3000), and 3000 ⁇ g of etoposide; in the step (4), 0.8 is added.
  • the polypeptide is as shown in SEQUENCE NO. 2 and 0.8 mg of the polypeptide as shown in SEQUENCE NO. 3 and 480 ⁇ g of EDC and 160 ⁇ g of NHS.
  • the targeted delivery system provided in Example 5 has a TEM particle size of 120-150 nm.
  • Example 6 A targeted delivery system and a preparation method are provided, which differ from the embodiment 1 in that 750 ⁇ g of doxorubicin in step (2) of Example 1 is replaced with 315 ⁇ g of abortion-type pregnancy drug - methotrexate whisper.
  • the targeted delivery system provided in Example 6 is a nano-sized spherical particle having an average particle size of 90-120 nm.
  • the encapsulation efficiency EN% of the targeted nanoparticles to methotrexate was 52.3 ⁇ 4.4%.
  • the abortion-type pregnancy drug in Example 6 can be replaced with at least one of mifepristone, misoprostol, letrozole, and prostamol, which can target the targeted delivery system.
  • mifepristone misoprostol
  • letrozole and prostamol
  • Example 7 A method for preparing a pl-CSA targeted delivery system (which can be a "targeted nanobubble"), which differs from Example 1 in that 750 ⁇ g of doxorubicin was replaced by step (2) of Example 1. 750g of the pregnancy drug - insulin growth factor 2; in step (4) is the amide reaction at 4 ° C; and step (3) is replaced by the following steps: (3) 1mL of PLGA acetonitrile solution to 0.3mL / The speed of min is added dropwise to the first mixed solution to obtain a second mixed solution; the second mixed solution is dissolved in a sealed 5 mL vial in an amount of 3 mL/bottle, and perfluoropropane is introduced into the vial. The air in the vial was set out and mechanically shaken for 2 min to obtain a drug-loaded suspension solution;
  • the above drug-suspended suspension solution was sonicated at a frequency of 20 kHz and a power of 130 W for 5 min, and the ultrasonicated solution was subjected to ultrafiltration centrifugation in an ultrafiltration centrifuge tube having a molecular weight cut-off of 10 KDa, and washed with PBS for 3 times. The supernatant was collected at a centrifugal speed of 3500 rpm for 3 min each to obtain a targeted delivery system precursor.
  • FIG. 3 is a schematic view showing the structure of a targeted nano delivery system prepared in Example 7 of the present invention.
  • the targeted nano delivery system differs from that of Figure 1 in that the target delivery comprises a gaseous contrast agent - perfluoropropane 121 and a non-abortionated pregnancy drug - insulin-like growth factor 2 (labeled 122), hydrophobic polymer PLGA11
  • the 121 and pregnancy drug 122 are wrapped and together with 121 and 122 form a hydrophobic core.
  • Example 8 A method for preparing a placenta-targeted delivery system differs from that of Example 7 in that in step (1), 2 mg of PLGA and 250 ⁇ g of liquid ultrasound contrast agent, perfluorooctyl ammonium bromide (PFOB), are dissolved.
  • PFOB perfluorooctyl ammonium bromide
  • a hydrophobic polymer solution was obtained in 1 mL of dichloromethane; in step (2), 40 ⁇ g of egg yolk lecithin, 100 ⁇ g of DSPE-PEG-NH 2 (having a molecular weight of PEG of 3000), and 250 ⁇ g of indomethacin Dissolved in 3 mL of acetone solution having a volume fraction of 4% to obtain a first mixed solution; in the step (3), 1 mL of the above hydrophobic polymer solution was added dropwise to the first mixed solution at a rate of 0.4 mL/min. In the middle, a drug-suspension suspension solution is obtained.
  • Figure 4 is a graph showing the particle size distribution of the targeted delivery system obtained in Example 8 of the present invention.
  • the targeted delivery system has an average particle size of 98 ⁇ 4.2 nm, wherein the number of particles below 180 nm accounts for 95.5%; and its PDI (Polymer Dispersibility Index) is 0.126 ⁇ 0.004, which indicates The prepared targeted delivery system has a relatively uniform particle size distribution.
  • Example 9 A method for preparing a placenta-targeted delivery system (which can be a "targeted nanobubble"), which differs from the embodiment 7 in that in the step (1), acetone is used as a solvent; in the step (2), The first mixed solution contains 800 ⁇ g of egg yolk lecithin, 1600 ⁇ g of DSPE-PEG-NH 2 (having a molecular weight of PEG of 3000), and 3000 ⁇ g of a relaxing peptide; in the step (3), the whole is introduced into the vial. Fluoropropane/nitrogen mixed gas (flow ratio is 1:1).
  • Figure 5 is a diagram showing the zeta potential distribution of the placenta-targeted delivery system obtained in Example 9 of the present invention.
  • the average zeta potential is -40.5 ⁇ 1 mV; and using a pH meter, the targeted delivery system has a pH of 6.5 ⁇ 0.2. Meets the criteria for intravenous injection.
  • Example 10 A preparation method of a targeted delivery system (which can be referred to as “targeted nanobubbles”) differs from Example 9 in that: "3000 ⁇ g of relaxed peptide" in step (2) of Example 9 is replaced with "750 ⁇ g of ELABELA polypeptide and 750 ⁇ g of relaxing peptide (this relaxing peptide was purchased from Nanjing Jinyibai Biotechnology Co., Ltd., item number JEB-10654)".
  • the prepared targeted nanobubbles contain a variety of pregnancy drugs.
  • FIG. 6 is a transmission electron micrograph of a targeted nanobubble obtained in Example 10 of the present invention.
  • the targeted nanobubbles have a spherical shape and a uniform size, and the particle diameter is about 80-100 nm; and the spherical color of each spherical nanobubble is a hydrophobic core, which is hydrophobic.
  • the core is PLGA and its coated ELABELA polypeptide, relaxin peptide and gaseous ultrasound contrast agent.
  • the pl-CSA targeted nano-delivery system (abbreviated as CSA-DNPs) prepared in Example 1 of the present invention was subjected to different cancer cell uptake experiments, and free doxorubicin (Free DOX) was used as a negative control, under the same conditions.
  • Peptide-grafted nanoparticles DNPs, the targeted delivery system precursors of the invention
  • SCR-DNPs scrambled polypeptide sets
  • the sequence of the scrambled polypeptide group SCR-DNPs modified polypeptide is as shown in SEQUENCE NO. 4: PNNKCESDKLAKHKKLGDKC, and the polypeptide has no targeting to placental-like chondroitin sulfate A.
  • JEG3 human placental chorionic cancer cells
  • 4T1 mouse breast cancer cells
  • RM1 mouse prostate cancer cells
  • HCC1937 human breast cancer cells
  • SKOV3 human ovarian cancer cells
  • free DOX group normal nanoparticle group (DNPs), scrambled peptide group (SCR-DNPs), placenta-like chondroitin sulfate A-targeted peptide group (CSA-DNPs)
  • DNPs normal nanoparticle group
  • SCR-DNPs scrambled peptide group
  • CSA-DNPs placenta-like chondroitin sulfate A-targeted peptide group
  • the cells were placed in a 37 ° C incubator for 30 min, and then the cells were washed 3 times with PBS buffer, then fixed with 4% paraformaldehyde for 20 min, washed with PBS 3 times, and added with 1 mL of 50 ⁇ g/mL DAPI solution. After standing at room temperature for 20 min, the cells were washed 4 times with PBS, and the cells of each group were observed by inverted fluorescence microscope. Light, the experimental results shown in Figure 7-8.
  • pl-CSA Highly expressed placenta-like chondroitin sulfate A
  • doxorubicin-coated pl-CSA-targeted nano delivery system provided by the present invention is surface-modified with a specific receptor for CSA (polypeptide LKPSHEKKNDDNGKKLCKAC),
  • the targeted nano delivery system is capable of rapidly (30 min) targeting to JEG3 cancer cells, which are taken up by cancer cells.
  • Figure 8 is a fluorescence imaging result of other cancer cells (4T1, RM1, HCC1937, SKOV3) treated with CSA-DNPs, wherein the first column and the second column correspond to those observed in the fluorescent channel of DAPI and doxorubicin, respectively. Fluorescent photograph, the third column is an overlay of the results of the first column and the second column. As can be seen from Fig. 8, the above other cancer cells were treated with CSA-DNPs, and strong red fluorescence was also observed (the red fluorescence corresponds to the lighter color in the second column of Fig. 4), which further verified the Targeting the targeting of nano delivery systems.
  • CSA-DNPs The doxorubicin-coated pl-CSA targeted nano-delivery system (abbreviated as CSA-DNPs) prepared in Example 1 of the present invention was tested for the effect of murine villus cancer, and was treated with PBS, free doxorubicin, ordinary nanoparticles, Scrambled polypeptide modified nanoparticles were used as controls.
  • the specific operations are as follows:
  • luciferase-labeled villous carcinoma cells Fluc-JEG3
  • PBS group free doxorubicin group
  • DNPs normal nanoparticle group
  • SCR-DNPs scrambled peptide Groups
  • CSA-DNPs doxorubicin-loaded pl-CSA targeted nano delivery systems
  • the equivalent of doxorubicin was 5 ⁇ g per injection in the other groups, and the tumor volume was recorded.
  • the tumor volume was calculated as: tumor length ⁇ (tumor width) 2 /2.
  • the experimental period is 18 days, and the test results are shown in Fig. 9.
  • Fig. 9 the PBS group can see obvious tumors, and the surrounding tissues are swollen, indicating that the tumor has spread and spread, and the tumors in the Free DOX group, the DNPs group, and the SCR-DNPs group appear black, but there is no spread. There were no visible tumors in the CSA-DNPs group compared with the other groups.
  • the test results show that the placenta-like chondroitin sulfate A targeted delivery system provided by the present invention can significantly inhibit the growth of cancer cells and has a good effect on the treatment of cancer.
  • CSA-IDNPs pl-CSA targeting nanoparticles
  • mice Female BALB/c nude mice weighing 4-20 weeks were used as test animals for 4-6 weeks, and nude mice were injected subcutaneously with 1 ⁇ 10 6 luciferase-labeled villous carcinoma cells (Fluc-JEG3), and passed after 5 days.
  • Fluc-JEG3 luciferase-labeled villous carcinoma cells
  • ICG equivalent 10 ⁇ g, and photographed under a small animal live imager at 10 min, 1 hour, and 31 hours, respectively, and the change of the fluorescence signal was recorded.
  • the test results are shown in FIG.
  • the left column of Figure 10 shows the location of luciferase-labeled villous carcinoma cells (Fluc-JEG3) (ie, tumor) in the IDNPs and CSA-IDNPs groups; the first row in the right column and the second row in the right column are injected with IDNPs.
  • Fluc-JEG3 luciferase-labeled villous carcinoma cells
  • CSA-IDNPs groups the first row in the right column and the second row in the right column are injected with IDNPs.
  • the fluorescence distribution of ICG in mice after CSA-IDNPs can be detected by small animal imager.
  • the tumor cells of the IDNPs group and the CSA-IDNPs group are mainly located in the right axillary region of the mouse.
  • the ICG fluorescence signal can be in the whole mouse.
  • Both the back and neck can be detected, but the fluorescent signal of ICG does not coincide well with the tumor cells in the axillary region, indicating that IDNPs did not reach the tumor site; after injection of CSA-DINPs, the right armpit of the mouse The tumor site is able to detect a strong fluorescent signal of ICG, indicating that CSA-DINPs have reached the tumor site, and the fluorescence signal can still be observed after 31 h, indicating that the coated nanoparticles coated with ICG can be quickly and permanently Targeting cancer cells can be used as an effective means to diagnose the location and development of cancer.
  • the pl-CSA-targeted nano-delivery system (abbreviated as CSA-MNPs) coated with methotrexate prepared in Example 6 of the present invention was tested for the effect of pregnant rats on abortion, and the PBS group and the free methotrexate group were used.
  • the normal nanoparticle group and the scrambled polypeptide group are used as a control, wherein the sequence of the polypeptide modified by the scrambled polypeptide group SCR-MNPs is as shown in SEQUENCE NO. 4, and the polypeptide has no targeting to the placenta.
  • mice Female CD-1 mice weighing 6-20 g at 6 weeks old were used as test animals, and female rats and male rats were caged at a ratio of 1:2. The female rats were examined for the next day, and the female shackles were seen. The day is 0.5 days of pregnancy.
  • Fig. 11 in the ultrasound development results on the 10th and 12th day, the reciprocal 2 images of each row are the experimental results of CSA-MNPs, showing the two different states of the pregnant mouse embryo after administration, the second to the bottom The figure shows that some pregnant mice have stunted growth, and the first mouse in the penultimate figure is close to death.
  • the CSA-MNPs group was able to see a large number of abortions (80% abortion) on the 14th day of pregnancy, and the embryo development was seen between the 9th and the 12th day of pregnancy compared to the PBS group. Slow, indicating that the placenta-targeted nano drug particles have impeded embryo growth and further prevented pregnancy.
  • the Free DOX group can also see abortion, but the number of abortions is limited to the 14th day of pregnancy, while the MNPs group, SCR- No abortion occurred in the MNPs group, and fetal growth was better.
  • the experiment was divided into a saline group and a targeted delivery system group.
  • the targeted delivery system prepared in the above Example 10 and the physiological saline were separately added to the latex gloves, solidified, and ultrapure water was added as an ultrasonic coupling agent in the beaker, and an ultrasonic diagnostic apparatus was used to set a superficial organ routine test mode.
  • Ultrasound imaging was performed on a glove equipped with a targeted delivery system and containing saline, and finally the stored images were collected. Among them, a glove group containing physiological saline was used as a negative control.
  • Figure 13 is an in vitro angiographic image of the saline group (a) and the targeted delivery system group (b).
  • In vitro ultrasound imaging can indirectly predict the effect of a placenta-targeted delivery system in vivo imaging.
  • the circle represents the finger part of the latex glove, which is filled with saline or nanobubbles. If the signal is black, it means there is no ultrasonic signal; if the circle is white with lighter color, it means There is an ultrasound signal in the area.
  • the results of Figure 13 show that the targeted delivery system has a strong contrast effect, and as the concentration increases, the ultrasound signal also increases.
  • the second pl-CSA targeted delivery system provided by the embodiments of the present invention has better targeting to tumor tissues or placental tissues expressing pl-CSA. It can be used in tumors, pregnancy diseases (including for abortion), and can be used in the diagnosis and treatment of other diseases related to pl-CSA expression.
  • the second targeted delivery system targeting pl-CSA and its preparation method are described below.
  • Example 11 A method of preparing a targeted delivery system comprising:
  • PLGA polylactic acid-glycolic acid copolymer
  • PFOB perfluorooctyl ammonium bromide
  • the nano-delivery system precursor is in an ultrafiltration centrifuge tube with a molecular weight cut-off of 10 kDa, washed with water, subjected to ultrafiltration centrifugation, and repeated three times, wherein the centrifugation speed is 36,000 rpm, each time of centrifugation for 4 minutes, and the supernatant is collected;
  • the above reaction solution was subjected to ultrafiltration centrifugation with a molecular weight cut-off 10 kDa ultrafiltration tube, and washed with PBS, and repeated 3 times, wherein the centrifugation speed was 3600 rpm, each centrifugation was performed for 3 min, and the supernatant was collected to obtain a placenta-like chondroitin sulfate A-targeting. Nano delivery system.
  • FIG 14 is a schematic view showing the structure of a placenta-like chondroitin sulfate A-targeted nano delivery system prepared in Example 11 of the present invention.
  • the targeted nano delivery system comprises a hydrophobic polymer layer 2', a viscous molecule 4' and a shell 3', wherein the viscous molecule 4' adheres to the surface of the hydrophobic polymer layer 2'
  • the outer shell 3' is an amphiphilic macromolecular compound 31' grafted to the polypeptide 32' of placenta-like chondroitin sulfate A, and the hydrophobic end of the amphiphilic macromolecular compound 31' is interspersed with the hydrophobic
  • the hydrophilic end of the amphiphilic macromolecular compound 31' is linked to the polypeptide 32' via an amide bond, and the polypeptide 32 is exposed outside the hydrophobic polymer layer 2, It is also exposed to the outermost surface of the targeted nano delivery system.
  • the targeted nano delivery system also includes a target delivery packaged by the hydrophobic polymer layer 2', the target delivery comprising the core of the targeted nano delivery system.
  • the target delivery includes a liquid ultrasound contrast agent PFOB (labeled 12' in the figure) and an antitumor drug doxorubicin 11'.
  • the component of the hydrophobic polymer layer 2 is PLGA, the viscous molecule 4 is PVA, and the amphiphilic macromolecular compound 31 is DSPE-PEG-COOH.
  • Example 12 A method of preparing a targeted delivery system, which differs from Example 1 in that:
  • step (1) 0.2 g of PLGA is dissolved in 4 mL of acetone, and 80 ⁇ L (5 mg/mL) of tetradecafluorohexane is added and stirred uniformly to obtain a PLGA solution; in the step (2), 80 mg of paclitaxel and 3 mg of DSPEG-NH2 is dissolved in 1 mL of acetonitrile to obtain a first mixed solution; in the step (7), 30 mL of a 2 wt% aqueous solution of PVA is added to the supernatant; in the step (8), 6 mg of the amide is added.
  • Figure 15 is a diagram showing the particle size distribution of the targeted nano delivery system obtained in Example 12 of the present invention.
  • the average particle diameter of the targeted nano-delivery system is 106 ⁇ 4.2 nm, wherein the distribution intensity of the nanobubbles below 200 nm reaches 82%; and the PDI (Polymer Dispersibility Index) is 0.126 ⁇ 0.004. This further demonstrates that the particle size distribution of the prepared targeted nano delivery system is relatively uniform.
  • Example 13 A method of preparing a targeted delivery system that differs from Example 1 in that:
  • step (1) 0.2 g of polycaprolactone is dissolved in 4 mL of chloroform, and 50 ⁇ L (6 mg/mL) of PFOB is added to obtain a polycaprolactone solution; in the step (2), 20 mg of methotrexate and 4 mg of DSPEG-COOH was dissolved in 1 mL of acetonitrile to obtain a first mixed solution; in the step (7), 30 mL of a 1.2% by weight aqueous solution of PVA was added to the supernatant, and in the step (8), when the amide reaction was carried out, A polypeptide as shown in SEQUENCE NO. 3, and 120 ⁇ g of EDC and 60 ⁇ g of NHS were added as a solvent in a MES buffer having a pH of 5.5.
  • FIG 16 is a transmission electron micrograph of a targeted nano delivery system obtained in Example 13 of the present invention.
  • the targeted nano-delivery system has a more regular spherical shape, a uniform size, and a particle size of about 100-120 nm.
  • the in vivo ultrasound capability study was performed on the pl-CSA targeted nano delivery system prepared in Example 11 of the present invention, and the non-targeted nano delivery system (unlinked polypeptide, ie, the drug-loaded nano delivery system precursor) was a non-targeted control group.
  • the specific operation is as follows:
  • FIG. 17 is a non-targeted nano delivery system group
  • FIG. 17 (b) is a target nano delivery system group
  • a circle represents a tumor.
  • the placenta-like chondroitin sulfate A was targeted to the nano-delivery system for 10 min, there was a strong ultrasound imaging signal at the tumor site, rather than a targeted nano-delivery system, and no ultrasound signal was detected at the tumor site. (when There is an ultrasonic signal, and the circle is white with a lighter color).
  • the targeted nano delivery system can be used as a novel targeted agent for the treatment and diagnosis of ovarian cancer. In addition, it can also be used in other diseases associated with pl-CSA.
  • a third targeted delivery system targeting pl-CSA and a method for its preparation are described below.
  • Embodiment 14 A method of preparing a targeted delivery system, comprising:
  • the above-mentioned targeted delivery system precursor was suspended in PBS solution, and the avidin-labeled polypeptide was added and incubated at room temperature (25 ° C) for 1-2 h; the obtained incubation solution was in an ultrafiltration centrifuge tube with a molecular weight cut off of 5 kDa.
  • the cells were centrifuged by ultrafiltration and washed with PBS for 3 times. Each ultrafiltration was centrifuged at 3000 rpm, and each ultrafiltration was centrifuged for 3 min. The supernatant after centrifugation was collected to obtain a placenta-like chondroitin sulfate A targeted delivery system.
  • FIG 18 is a schematic view showing the structure of a placenta-like chondroitin sulfate A targeted delivery system prepared in Example 14 of the present invention.
  • the targeted delivery system comprises a serum albumin layer 2" and a carbohydrate molecule 3", wherein the carbohydrate molecule 3" is adhered to the serum albumin layer 2", and the serum albumin layer 2" is also grafted with a target Polypeptide 4" of placental-like chondroitin sulfate A, one end of polypeptide 4" is labeled with avidin 41", and the outer surface of serum albumin layer 2" is adsorbed with biotin 21", such that polypeptide 4" passes through avidin The specific binding force between protein 41" and biotin 21" is grafted onto the serum albumin layer 2".
  • the targeted delivery system further includes a target delivery package wrapped by the serum albumin layer 2", the target delivery material forming the core of the targeted delivery system.
  • the target delivery comprises a gaseous ultrasound contrast agent PFOB (labeled in the figure is 12"), and the antitumor drug methotrexate 11", the carbohydrate molecule 3" is specifically glucose.
  • the mass ratio of the serum albumin to the saccharide molecule is 1:2; the mass ratio of the serum albumin to the polypeptide is 1:5; and the mass ratio of biotin to serum albumin is 1:1000.
  • Example 15 A method of preparing a targeted delivery system that differs from Example 14 in that:
  • PBS isotonic phosphate buffer
  • the mass ratio of the serum albumin to the carbohydrate molecule is 1:2.4; the mass ratio of serum albumin to polypeptide is 1:3.2; the quality of biotin and serum albumin The ratio is 1:625.
  • the encapsulation efficiency of the methotrexate in the targeted delivery system was EN ⁇ 56 ⁇ 3.2%, and the drug loading rate was 2.56 ⁇ 0.37%.
  • the targeted delivery system prepared in Example 14 was added to the adherent cultured breast cancer MDA-MB-231 cells in an amount of 1 ⁇ 10 8 microbubbles/mL as a targeted drug-loaded microbubble treatment group.
  • the following sets of experiments were also set up: a completely untreated group (ie, PBS buffer), an unloaded microbubble group (ie, a targeted delivery system without methotrexate), a non-targeted drug-loaded microbubble group (That is, the unmodified polypeptide targets the drug-loaded microvesicle precursor).
  • the cells of the above four treatment groups were treated with sonication and non-sonication respectively. After 24 hours of culture, the survival rate of the cells was analyzed by CCK-8 method, and the specific killing activity of microvesicles on breast cancer cells under ultrasonic conditions was analyzed. As shown in Figure 19.
  • the targeted drug-loaded ultrasonic microbubbles have stronger killing activity than the non-targeted drug-loaded ultrasonic microbubbles, and reflect better breast cancer therapeutic effects.
  • Breast cancer is a disease associated with inappropriate expression of pl-CSA, and the above results indicate that the pl-CSA targeted delivery system provided by the present invention can be used as a treatment and diagnosis for other diseases associated with pl-CSA.
  • the fourth targeted delivery system targeting pl-CSA and its preparation method are described below.
  • Example 16 A method of preparing a pl-CSA targeted delivery system comprising:
  • the reaction solution was centrifuged at 10,000 rpm for 6 min, the supernatant was discarded, and the precipitate was collected, followed by washing with deionized water, and repeated 4 times to obtain a drug carrier, that is, an oxidized carbon nanotube covalently linked to the polypeptide.
  • doxorubicin 2a is an oxidized carbon nanotube 11a covalently linked to a polypeptide 12a that targets placenta-like chondroitin sulfate A.
  • doxorubicin 2a is bonded to the surface of the drug carrier by physical adsorption, and more specifically, adsorbed on the oxidized carbon nanotube 11a by a ⁇ - ⁇ force.
  • the mass ratio of the oxidized carbon nanotubes 11a to the polypeptide 12a is 3:20, that is, 1:6.67; and the mass ratio of the drug carrier to the doxorubicin 2a is 1:3.
  • Example 17 A preparation method of a pl-CSA targeted delivery system, which differs from Example 16 in that, in the preparation of the oxidized carbon nanotubes, a single wall of 100 mg and a diameter of 10 nm is used in the step (1).
  • Carbon nanotubes; in step (2), 20 mg of oxidized carbon nanotubes were added to 100 mL of MES buffer (pH 5.5), 0.15 g of NHS and 0.15 g of EDC were added, activated for 2 h, and then 0.25 g of The polypeptide represented by SEQUENCE NO.1 was subjected to amidation reaction at room temperature for 24 hours to obtain a reaction solution; in the step (3), the mass of the drug carrier was 5 mg, and the mass of doxorubicin was 20 mg.
  • the mass ratio of the oxidized carbon nanotubes to the polypeptide is 2:25, that is, 1:12.5; the mass ratio of the drug carrier to the doxorubicin is 1:4.
  • Figure 21 is a transmission electron micrograph of the prepared pl-CSA targeted delivery system, as seen from Figure 21, the length of the carbon nanotubes is about 150 nm.
  • Example 16 2 mg of the lyophilized powder of the targeted delivery system prepared in Example 16 was accurately weighed and dispersed in phosphate buffers of pH 7.4, pH 6.5 and pH 5.5, respectively, to obtain a targeted delivery system at a concentration of 1 mg/ mL of sample solution. 1 mL of the sample solution was placed in a dialysis bag with a molecular weight cutoff of 3,500, and the dialysis bags were placed in a beaker containing 100 mL of the corresponding phosphate buffer solution, and placed in a 37 ° C constant temperature oscillator for dialysis, respectively, at 1 h.
  • the in vitro release of the pl-CSA targeted delivery system loaded with doxorubicin hydrochloride is pH dependent, and the lower the pH, the higher the cumulative release and release rate of doxorubicin, indicating the present invention.
  • the provided pl-CSA targeted delivery system is relatively stable under physiological conditions and is released more under acidic conditions of tumor tissue.
  • Human lung cancer cell A549 cells in logarithmic growth phase were inoculated into a 6-well plate at a density of 105/mL, and 2 mL of DMEM medium was added to each well, and cultured at 37 ° C for 24 h, and the number of cells increased by 50%-70%. Thereafter, the medium in the well was replaced with 2 mL of DMEM drug medium containing a delivery system without a polypeptide, 2 mL of a DMEM drug medium containing a polypeptide-binding delivery system (prepared in Example 14), and incubated at 4 ° C.
  • the uptake rate of cells to the pl-CSA targeting group loaded with doxorubicin was 86 ⁇ 2.2%, while the uptake rate for the non-targeted group of unlinked polypeptides was only 5.2 ⁇ 1.6%.
  • the above significant differences indicate that modification of the carbon nanotubes in the delivery system with a polypeptide that targets pl-CSA significantly increases the specific uptake of the delivery system by the cells.
  • FIG. 24 is a schematic diagram showing the synthesis of a fifth pl-CSA targeted delivery system, which may also be referred to as a polypeptide-drug conjugate, according to an embodiment of the present invention.
  • 1b is a small molecule drug
  • 2b is a linker
  • 3b is a polypeptide targeting pl-CSA, which are linked together by a chemical bond to obtain a polypeptide-drug conjugate targeting pl-CSA.
  • the resulting polypeptide-drug conjugate targeting pl-CSA comprises a small molecule drug residue 1b', a polypeptide 3b' residue, and a linker joining the small molecule drug residue 1b', the polypeptide residue 3b' group 2b ', wherein the polypeptide residues 3b' which targets pl-CSA polypeptide or remove part of the -NH 2 -COOH.
  • the small molecule drug residue 1' refers to a residual portion of the small molecule drug 1 after removing an active group that does not affect its drug activity, and its overall structure is very close to that of the small molecule drug 1.
  • the linker group 2' may be similar to the structure of the linker 2, and may also be quite different, especially when the linker 2 is a cyclic dianhydride.
  • a polypeptide-drug conjugate targeting placenta-like chondroitin sulfate A (pl-CSA): firstly reacting doxorubicin hydrochloride with succinic anhydride, introducing a carboxyl group into the doxorubicin molecule; The doxorubicin is subjected to an amide reaction with the polypeptide to react the terminal amino group of the polypeptide fragment with the carboxyl group on the carboxylated doxorubicin to form a targeting polypeptide-drug conjugate.
  • the reaction equation is as follows:
  • a polypeptide-drug conjugate targeting pl-CSA firstly reacting paclitaxel with succinic anhydride to introduce a carboxyl group on the paclitaxel molecule; and then subjecting the carboxylated paclitaxel to the amide reaction of the polypeptide to make the end of the polypeptide fragment
  • the amino group reacts with a carboxyl group on the above carboxylated paclitaxel to form a polypeptide-drug conjugate.
  • the preparation process includes the following steps:
  • Example 20 A method for preparing a polypeptide-drug conjugate targeting pl-CSA, which differs from Example 18 in that: Example 20 is replaced by 0.1 g of a polypeptide of SEQUENCE NO. 2 (EDVKDINFDTKEKFLAGCLIVSFHEGKC). The polypeptide used in Example 18.
  • the choriocarcinoma JEG3 cells were used as the research object, and the antitumor effect of the targeted polypeptide-drug conjugate provided by the present invention was evaluated by the MTT method. details as follows:
  • Collect log phase cells adjust the cell suspension concentration, add 100 ⁇ L per well, and plate the cells to adjust the density to 1000-10000 per well. Incubate at 5% CO2 at 37 °C until the cell monolayer is filled with the bottom of the well (96-well flat bottom plate). After 4 hours, add 7 concentration gradients (0, 0.2, 0.4, 0.6, 0.8, 1, 2).
  • Free DOX Free DOX
  • DOX-plCSA targeting polypeptide-drug conjugate
  • the well culture solution was carefully aspirated, 150 ul of dimethyl sulfoxide (DMSO) was added to each well, and shaken on a shaker at low speed for 5 min to dissolve the crystals sufficiently.
  • the absorbance value A of each well was measured at an enzyme-linked immunosorbent detector OD490nm.
  • zero-adjusting wells medium, MTT, DMSO
  • control wells cells, the same concentration of drug dissolution medium, culture solution, MTT, DMSO
  • Proliferation inhibition rate 1 - (experimental hole A value - zero hole A value) / (control hole A value - zero hole A value), wherein the actual A value of each group is the result after subtracting the withering hole.
  • Fig. 25 The results of the inhibition rate of proliferation of JEG3 cells by each experimental group are shown in Fig. 25, wherein * indicates P ⁇ 0.05 compared with the PBS group; ** indicates P ⁇ 0.01 compared with the PBS group.
  • the experimental results in Figure 25 show that the conjugate DOX-plCSA has a strong antitumor effect compared to free doxorubicin (Free DOX).
  • the fifth targeted delivery system provided by the present invention has high degree of targeting and enrichment for tissues that improperly express pl-CSA, and can effectively improve the drug effect of small molecule drugs.

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Abstract

La présente invention concerne un polypeptide ciblant le sulfate A de chondroïtine placentaire, un système d'administration ciblée ciblant le sulfate A de chondroïtine placentaire, et un procédé de préparation. Le polypeptide et le système d'administration ciblée fournis par la présente invention sont hautement ciblés vers un tissu exprimant le sulfate A de chondroïtine placentaire. La présente invention concerne également des applications du polypeptide et du système d'administration ciblée.
PCT/CN2017/108646 2017-09-28 2017-10-31 Polypeptide ciblant le sulfate a de chondroïtine placentaire, système d'administration ciblée, son procédé de préparation et son application WO2019061648A1 (fr)

Applications Claiming Priority (14)

Application Number Priority Date Filing Date Title
CN201710906204.5 2017-09-28
CN201710906586.1 2017-09-28
CN201710903031.1 2017-09-28
CN201710905200.5A CN109568598B (zh) 2017-09-28 2017-09-28 用于药物流产的胎盘靶向纳米颗粒及其制备方法和应用
CN201710905199.6A CN109589416B (zh) 2017-09-28 2017-09-28 胎盘样硫酸软骨素a靶向纳米投递系统及其制备方法和应用
CN201710903483.X 2017-09-28
CN201710906204.5A CN109568596B (zh) 2017-09-28 2017-09-28 胎盘样硫酸软骨素a靶向运载体系及其制备方法和应用
CN201710905200.5 2017-09-28
CN201710906587.6A CN109589413B (zh) 2017-09-28 2017-09-28 靶向胎盘样硫酸软骨素a的多肽、靶向纳米颗粒及其制备方法和应用
CN201710906587.6 2017-09-28
CN201710903483.XA CN109568289B (zh) 2017-09-28 2017-09-28 胎盘样硫酸软骨素a靶向传输系统及其制备方法和应用
CN201710905199.6 2017-09-28
CN201710906586.1A CN109568597B (zh) 2017-09-28 2017-09-28 靶向胎盘样硫酸软骨素a的多肽药物偶联物及其制备方法和应用
CN201710903031.1A CN109568268A (zh) 2017-09-28 2017-09-28 胎盘靶向递送系统及其制备方法和应用

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Publication number Priority date Publication date Assignee Title
WO2022016375A1 (fr) * 2020-07-21 2022-01-27 深圳先进技术研究院 Polypeptide de reconnaissance de l'endomètre ectopique, dérivé et utilisation associés
WO2022016752A1 (fr) * 2020-07-21 2022-01-27 深圳先进技术研究院 Système de nano-libération ciblant les lésions de l'endométriose, son procédé de préparation et son utilisation
CN114085807A (zh) * 2021-09-24 2022-02-25 复旦大学附属妇产科医院 胎儿滋养层细胞捕获探针及其用途

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CN103040757A (zh) * 2012-12-26 2013-04-17 深圳先进技术研究院 核-壳型纳米药物颗粒、其制备方法及应用
CN103099782A (zh) * 2012-08-10 2013-05-15 深圳先进技术研究院 核-壳型纳米药物颗粒、其制备方法及应用
CN104136041A (zh) * 2012-02-09 2014-11-05 Var2制药有限公司 靶向硫酸软骨素聚糖
WO2016135291A1 (fr) * 2015-02-26 2016-09-01 Var2 Pharmaceuticals Aps Ciblage immunothérapeutique de sulfate de chondroïtine de type placentaire à l'aide de récepteurs d'antigènes chimériques (car) etciblage immunothérapeutique du cancer à l'aide de car avec des systèmes de liaison de protéines de division

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CN104136041A (zh) * 2012-02-09 2014-11-05 Var2制药有限公司 靶向硫酸软骨素聚糖
CN103099782A (zh) * 2012-08-10 2013-05-15 深圳先进技术研究院 核-壳型纳米药物颗粒、其制备方法及应用
CN103040757A (zh) * 2012-12-26 2013-04-17 深圳先进技术研究院 核-壳型纳米药物颗粒、其制备方法及应用
WO2016135291A1 (fr) * 2015-02-26 2016-09-01 Var2 Pharmaceuticals Aps Ciblage immunothérapeutique de sulfate de chondroïtine de type placentaire à l'aide de récepteurs d'antigènes chimériques (car) etciblage immunothérapeutique du cancer à l'aide de car avec des systèmes de liaison de protéines de division

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022016375A1 (fr) * 2020-07-21 2022-01-27 深圳先进技术研究院 Polypeptide de reconnaissance de l'endomètre ectopique, dérivé et utilisation associés
WO2022016752A1 (fr) * 2020-07-21 2022-01-27 深圳先进技术研究院 Système de nano-libération ciblant les lésions de l'endométriose, son procédé de préparation et son utilisation
CN114085807A (zh) * 2021-09-24 2022-02-25 复旦大学附属妇产科医院 胎儿滋养层细胞捕获探针及其用途

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