WO2016041250A1 - 一种炎症靶向的中性粒细胞递药系统及其应用 - Google Patents

一种炎症靶向的中性粒细胞递药系统及其应用 Download PDF

Info

Publication number
WO2016041250A1
WO2016041250A1 PCT/CN2014/092340 CN2014092340W WO2016041250A1 WO 2016041250 A1 WO2016041250 A1 WO 2016041250A1 CN 2014092340 W CN2014092340 W CN 2014092340W WO 2016041250 A1 WO2016041250 A1 WO 2016041250A1
Authority
WO
WIPO (PCT)
Prior art keywords
inflammation
delivery system
drug
neutrophil
inflammatory
Prior art date
Application number
PCT/CN2014/092340
Other languages
English (en)
French (fr)
Inventor
张灿
薛敬伟
赵泽恺
张蕾
温雅静
Original Assignee
中国药科大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国药科大学 filed Critical 中国药科大学
Priority to US15/512,726 priority Critical patent/US11085022B2/en
Publication of WO2016041250A1 publication Critical patent/WO2016041250A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0642—Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells
    • 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
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5383—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0097—Cells, viruses, ghosts, red blood cells, viral vectors, used for imaging or diagnosis in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1896—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes not provided for elsewhere, e.g. cells, viruses, ghosts, red blood cells, virus capsides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1203—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules in a form not provided for by groups A61K51/1206 - A61K51/1296, e.g. cells, cell fragments, viruses, virus capsides, ghosts, red blood cells, viral vectors
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention belongs to the field of pharmaceutical preparations, and relates to an inflammatory targeted neutrophil delivery system and application thereof, and particularly to a delivery system for targeting an inflammation site using neutrophils as a carrier.
  • Inflammation refers to the physiological response of biological tissues that are stimulated by trauma, hemorrhage, or pathogen infection, including redness, fever, and pain.
  • the inflammatory response is an important defense mechanism for the innate immune system to remove harmful stimuli or pathogens and promote repair. According to the cause of the disease, it is divided into infectious inflammation, non-specific inflammation and allergic inflammation.
  • Infectious inflammation is inflammation caused by viruses, bacteria or bacterial products, usually treated with antibiotics or antifungal drugs; non-specific inflammation is the use of certain proinflammatory factors to cause inflammation or a certain stage of cellular tissue in animals or in vitro.
  • Reactions such as ischemia-reperfusion injury, postoperative injury, etc., require systemic therapy; allergic inflammation is the terminal manifestation of polymorphic allodynia, such as lupus dermatitis, which requires anti-immunotherapy.
  • the pathological features of inflammation mainly include local tissue deterioration, exudation and hyperplasia. Due to the high permeability of the inflammatory blood vessels, the protein-rich liquid and leukocytes in the blood vessels exude, resulting in an increase in the interstitial pressure of the local tissue of the inflammation. It can be seen that the high permeability of blood vessels at the site of inflammation provides a possibility for the drug to escape from the blood vessels and stay in the site of inflammation, but the higher interstitial pressure of the inflammation tissue hinders the entry of the drug into the site of inflammation.
  • Targeted delivery systems fall into three broad categories: passive targeting, active targeting, and physicochemical targeting. Compared with the previous delivery systems, although these emerging targeted delivery systems have made a qualitative leap, there are still shortcomings in targeting, strong side effects, and the diagnosis and treatment effects are difficult to achieve the expected shortcomings. Therefore, it is extremely necessary to find A more efficient, less toxic targeted delivery system.
  • Immune cells including mononuclear phagocytic cells (dendritic cells, monocytes and macrophages), neutrophils and lymphocytes cell.
  • the exudation of immune cells during inflammation is the most important feature of inflammatory response, and neutrophils, as "defense guards" when inflammation occurs, are attracted by chemokines to reach the site of inflammation for the first time.
  • neutrophils as "defense guards" when inflammation occurs, are attracted by chemokines to reach the site of inflammation for the first time.
  • no neutrophils have been seen as vectors for the same or similar reports of inflammatory site drug, probe and developer delivery studies.
  • There are some scientific problems to be solved in the loading of drugs or nano-agents into neutrophils such as the way drugs are loaded, the amount of cells loaded with drugs, probes and developers, the stability of the loaded drugs in cells, and the inherent physiology of cell carriers. Problems such as maintenance of activity.
  • the present invention provides a neutrophil delivery system for a drug that targets inflammatory tissue.
  • Another object of the invention is to provide an application of the delivery system.
  • An inflammatory targeted neutrophil delivery system consisting of neutrophils and therapeutic or detection substances that are loaded directly or indirectly into neutrophils or surfaces.
  • the therapeutic substance includes, but is not limited to, one or more of a drug (a chemical synthetic drug, a natural drug or a natural drug extract), a therapeutic DNA, an RNA, a protein or a polypeptide; the detection The substance is selected from one or more of a probe and a developer.
  • a drug a chemical synthetic drug, a natural drug or a natural drug extract
  • a therapeutic DNA an RNA, a protein or a polypeptide
  • the indirect manner refers to using the nanocarrier as a tool, loading a therapeutic substance or a detecting substance into the nano carrier first, forming a nano preparation, and then loading the nano preparation into the neutrophil or the surface.
  • the nanocarriers are preferably self-positive, electronegative or near-neutral nanoparticle preparations having a particle size of from 1 to 1000 nm.
  • the nanocarriers include, but are not limited to, gold nanoparticles/rods, magnetic nanoparticles, mesoporous silica nanoparticles, graphene, liposomes, micelles, nanoemulsions, nanospheres, nanocapsules, microspheres, micro Pills or dendrimers.
  • the drug is selected from one or more of an efferent nervous system drug, a central nervous system drug, a cardiovascular system drug, a hormone and a respiratory digestive system drug, an antibacterial or antiviral drug, an antitumor drug, and a vitamin.
  • the drug is preferably a self-antipyretic analgesic drug, a non-steroidal anti-inflammatory drug, an anti-tumor drug, an antibiotic drug, a diuretic, an antihypertensive drug, a lipid-lowering drug, a hypoglycemic agent, a hormone drug or a vitamin.
  • a self-antipyretic analgesic drug preferably a self-antipyretic analgesic drug, a non-steroidal anti-inflammatory drug, an anti-tumor drug, an antibiotic drug, a diuretic, an antihypertensive drug, a lipid-lowering drug, a hypoglycemic agent, a hormone drug or a vitamin.
  • a self-antipyretic analgesic drug preferably a self-antipyretic analgesic drug, a non-steroidal anti-inflammatory drug, an anti-tumor drug, an antibiotic drug, a diuretic, an antihypertensive drug, a lipid-lowering drug, a hypog
  • the drug is further preferably cetorus, phenytoin, chlorpromazine, fluoxetine, methadone, meclofenoxate, chlorofeline, atropine sulfate, isoproterenol, chlorpheniramine maleate, hydrochloric acid.
  • inflammatory targeted neutrophil delivery system of the present invention in the manufacture of a medicament or agent for treating or diagnosing an inflammation and/or inflammation related disease.
  • the inflammation includes infectious inflammation, non-specific inflammation, allergic inflammation, and inflammation-related diseases.
  • the infectious inflammation includes inflammation caused by a virus, a bacterium or a bacterial product; the non-specific inflammation includes physical inflammation, including redness, pain, etc. caused by surgery or trauma; allergic inflammation including lupus dermatitis, allergy Asthma, rheumatoid arthritis; inflammation-related diseases include treatment of postoperative recurrence of tumors, atherosclerosis, hypoxic ischemic encephalopathy.
  • the invention coats the therapeutic substance or the detecting substance in different electric nano carriers (positive, negative, near-neutral, 1-1000 nm), and then loads the drug-loaded nano carrier (ie, nano preparation)
  • Neutron granule-loaded delivery systems have been successfully prepared by loading/producing neutrophils/surfaces; or directly loading therapeutic substances or detection substances into neutrophils/surfaces.
  • the prepared neutrophil delivery system is intravenously injected into the human body, and during the blood circulation, it is stimulated by chemokines released from the inflammatory region, passes through the blood vessels of the inflammation site, and is actively positioned along the concentration gradient of the chemokine.
  • the site of inflammation increases the concentration of the drug in the inflammation site and ensures the efficacy of the drug.
  • neutrophils are used as a delivery system to treat inflammation or Inflammation-related diseases such as tumor recurrence after surgery using acute inflammation can effectively increase the accumulation of drugs at the target site, improve the drug's efficacy, and reduce its side effects.
  • the neutrophils loaded with the therapeutic substance or the detecting substance can be successfully delivered to the inflammation-related disease site by the inflammatory factor, and the disease can be treated and diagnosed. Inhibit the occurrence and development of the disease.
  • a medicament for treating inflammation and/or inflammation related diseases comprising the inflammatory targeted neutrophil delivery system of the present invention.
  • the therapeutic substance in the neutrophil delivery system may be a drug having a therapeutic effect on inflammation and/or inflammation-related diseases in a conventional administration route, including but not limited to a nervous system drug, a cardiovascular and cerebrovascular drug. , blood, respiratory, digestive system drugs, endocrine system and immunomodulatory drugs such as immunopotentiating drugs interferon, immunosuppressive cyclosporine.
  • An agent for diagnosing an inflammation and/or inflammation related disease comprising the inflammatory targeted neutrophil delivery system of the invention.
  • the diagnostic substances in the neutrophil delivery system include, but are not limited to, probes such as DNA probes, fluorescent probes, contrast agents such as X-ray observations of commonly used iodine preparations, barium sulfate, and nuclear magnetic imaging contrast agents such as sputum. Spray acid glucamine and so on.
  • Nanocarriers nanoscale drug carriers, a submicron drug carrier delivery system belonging to the nanoscale microscopic category. Encapsulation of the drug in the submicron (ie, the nanocarrier) can adjust the rate of drug release, increase the permeability of the biofilm, change the distribution in the body, and improve bioavailability.
  • nanoformulations of the present invention can be prepared according to any of the methods reported in the prior art.
  • Liposomes suitable for the preparation of fat-soluble drugs include a film dispersion method, an injection method, a precursor liposome method, an ultrasonic dispersion method, and the like.
  • this method includes reverse phase evaporation, double emulsion method, melting method, freeze-thaw method, freeze-drying method, surface activity.
  • Methods such as a treatment method, a calcium fusion method, and a centrifugation method.
  • Inflammation The defense response of the living tissue with the vascular system to the injury factor is inflammation.
  • Neutrophils are the most abundant white blood cells, accounting for 50% to 70% of white blood cells, and are easily obtained from patients. This provides a rich reserve for drug delivery.
  • the invention uses neutrophils as a carrier for delivery.
  • the neutrophil protects the drug, probe or developer contained from the reticuloendothelial system, prolongs the half-life, effectively controls the release, and reduces the immunogenicity and toxicity. side effect.
  • neutrophils can be used as a carrier to smoothly reach the affected area through the hydrophobic barrier and increase the concentration of the target site.
  • neutrophils because neutrophils have chemotactic properties, they can be targeted to lesions, inflammation, and tumor site release. Therefore, neutrophils, as low-toxic transporters, can serve as targeted delivery, prolong cycle times, and reduce the toxicity of cells and tissues.
  • neutrophils can be used as a carrier of drugs. Actively target the drug to the site of inflammation and increase the concentration of the drug at the site of inflammation.
  • the neutrophils reaching the inflammation site are abnormally activated under the stimulation of cytokines, and the rapid disintegration is killed in the form of "Skynet", which is beneficial to the rapid release of the loaded drug to the target site, improving the therapeutic effect and reducing the side effects.
  • the present invention has the following advantages:
  • the neutrophil delivery system successfully prepared by the invention reaches the inflammatory tissue with a concentration gradient of chemokines, and successfully solves the targeting problem of drugs, probes or developers.
  • the neutrophil delivery system prepared by the invention ensures the high drug loading of neutrophils by mediated by the nanocarrier, and avoids the influence of the drug on the physiological activity of neutrophils.
  • the neutrophil delivery system prepared by the present invention since neutrophils are a natural immune cell of the body, and have strong phagocytosis, the amount of drugs, probes or developers contained in the cells is high.
  • the drug carrier neutrophil used in the present invention is the most abundant immune cell in the human body, and is easily obtained from a patient, and the neutrophil is lysed by a unique death mode after abnormal activation of the inflammation site.
  • the drug is quickly and completely released into the inflamed tissue, which makes the invention have high clinical development and application value.
  • Fig. 5 Relative ingestion rate of the neutrophil delivery system of the negatively charged, near-neutral, and positively charged nano-preparation of the post-operative glioma model mice relative to the commercially available preparation.
  • Figure 6 Targeting efficiency of neutrophil delivery system for commercially available formulations and load negatively charged, near-neutral, positively charged nanoformulations after administration of mice.
  • Figure 7 The cerebral glioma postoperative model mice were administered with physiological saline, blank neutrophils, commercial formulations of Taxol, load-negative, near-neutral, positively charged nano-preparative neutrophil delivery system. Period curve.
  • Figure 9 Relative uptake rate of inflammatory neutrophil delivery system relative to commercial formulations after administration of pneumococcal pneumonia model mice.
  • FIG. 11 Survival curve of normal saline, blank neutrophils, commercial levofloxacin and drug-loaded neutrophil delivery system in mice with pneumococcal pneumonia model.
  • Figure 13 Relative uptake rate of inflammatory site neutrophil delivery system relative to commercial formulations after administration of ear swelling model mice.
  • FIG. 15 Ear swelling model mouse mice were administered with physiological saline, blank neutrophils, commercial preparations of ibuprofen, and drug-borne neutrophil delivery system ear swelling degree.
  • Figure 17 Relative uptake rate of inflammatory site neutrophil delivery system relative to commercial formulations after administration of adjuvant arthritis model mice.
  • Fig. 19 Adjuvant arthritis model mice were administered with physiological saline, blank neutrophils, a commercial preparation of ibuprofen, and a drug-loaded neutrophil delivery system arthritis index score.
  • Example 1 Targeting and pharmacodynamic evaluation of paclitaxel liposome neutrophil delivery system on postoperative glioma mouse model with different surface potentials
  • Percoll stock solution was prepared as a 100% Percoll at 9:1 (v/v) with 10 x PBS, and diluted to 15% PBS to prepare a 55%, 65%, 68% (v/v) Percoll separation.
  • the 55% and 65% two-layer Percoll separation liquid was prepared by slowly and uniformly superimposing, and the freshly extracted mouse tibia bone marrow cells were diluted with 1 ⁇ PBS to prepare a single cell suspension, 1: 1 (v/v) was slowly added uniformly to the top of the 55%, 65% Percoll separation solution and centrifuged at 1000 g for 30 min.
  • Bone marrow cells with 55% and 65% separation solution were extracted, washed in one volume of 1 ⁇ PBS and resuspended, then slowly superimposed on 68% Percoll separation solution and centrifuged at 1000g for 30min, and the bone marrow cells of 68% separation layer were extracted and added.
  • the mouse bone marrow neutrophil suspension was prepared by washing one time in 1 ⁇ PBS, repeated twice, and resuspended in RPMI1640 medium.
  • the extracted cells were stained with a red fluorescently labeled cell surface protein Gr-1 antibody (Beijing Dakco Biotech Co., Ltd.); the cells were incubated at 37 ° C, 5% CO 2 for 30 min, and the extracted cells were examined by flow cytometry. The activity of the cells was determined to be about 98% by using trypan blue. The morphological purity of the extracted cells was identified by Wright-Gemsa staining. The results are shown in Figure 1. Most of the neutrophil nuclei were isolated. It was mature or lobulated; the extracted cells were stained with the red fluorescently labeled cell surface protein Gr-1 antibody, and the purity of the extracted neutrophils was found to be higher than 90%.
  • Gr-1 antibody Beijing Dakco Biotech Co., Ltd.
  • Negative liposome soybean phospholipid 100mg + cholesterol 10mg + paclitaxel 5mg;
  • each substance was dissolved in a mixed solvent of chloroform/methanol (2:1, v:v), and after mixing, it was vacuum-dried into a film by a vacuum bath at 40 ° C, and placed in a vacuum desiccator overnight to completely remove the organic solvent. After that, the lipid film was hydrated at 37 ° C with ultrapure water, and the obtained lipid suspension was dispersed in an ultrasonic cell powder ice bath, and passed through a 0.45 ⁇ m and 0.22 ⁇ m microporous membrane to obtain three different electrical properties.
  • Paclitaxel loaded liposomes PTX-Liposome
  • the drug loading and encapsulation efficiency of PTX-Liposome were measured by HPLC, and the particle size and potential were measured using a laser particle size analyzer as shown in Table 1.
  • the freshly prepared neutrophil suspension was inoculated into a 24-well plate at 1 ⁇ 10 6 cells/well and 500 ⁇ L.
  • the serum-free RPIM1640 medium was cultured at 37° C. and 5% CO 2 for 1 h, and the culture solution was removed.
  • Three different electrical paclitaxel liposomes were diluted into PTX 100 ⁇ g/ml with serum-free RPIM1640 medium and added to the above 24-well plate. After incubation at 37 ° C for 50 min, the culture medium containing the preparation was discarded and washed three times with 4° C. PBS. That is, a paclitaxel liposome-neutral cell delivery system with different surface potentials.
  • Ph7.4RPMI1640 medium, 10nM chemotactic tripeptide and 100nM phorbol alcohol were used to simulate three physiological and pathological conditions of normal physiological conditions, chemotaxis process and inflammation site, respectively.
  • Four kinds of electrical paclitaxel liposomes were determined by HPLC. The retention in neutrophils under different physiological and pathological conditions, the results are shown in Figure 4. The results showed that the cumulative drug release in the process of chemotaxis only accounted for 5% of the total intake, and the release was slow, and reached the target site. Under the abnormal activation of neutrophils, the cumulative drug release accounted for the total intake. 85% of the time, to ensure that most of the drugs can reach the target site and play a role.
  • mice were anesthetized with chloral hydrate by intraperitoneal injection and fixed on a stereotactic head frame.
  • Mouse glioma cell G422 was injected into the right caudate nucleus of mice with a micro syringe (surgical exposure of the skull marker, with a dental drill
  • the front sac is 4mm apart, the front 1mm, the aperture is 1.2mm, and the subdural needle is 5mm deep. Stitch, disinfect and put back in the cage.
  • Postoperative glioma model mice 144, were randomly divided into 4 groups, 36 in each group. The rats were fasted to avoid overnight water before administration.
  • the 4 groups of tumor-bearing mice were intravenously injected with paclitaxel, a commercial preparation of Taxol (Taxol).
  • paclitaxel a commercial preparation of Taxol (Taxol).
  • PTX dose is 5mg/kg, respectively 0.167h, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h after administration
  • At 48h, 72h, 96h and 120h 3 tumor-bearing mice were taken from each group. After taking the eyeballs and taking blood, the mice were sacrificed.
  • tissue samples of heart, liver, spleen, kidney and brain were taken and washed with physiological saline. And weighed the residual physiological saline with a filter paper and weighed. Each tissue was treated as follows, and the concentration of PTX contained in each tissue sample at each time point was measured by an HPLC method.
  • Tissue samples (heart, liver, spleen, lung, kidney, brain) of tumor-bearing mice were weighed and placed in blood collection tubes. 2 mL of physiological saline was added and dispersed into a tissue homogenate by a tissue homogenizer at high speed. 200 ⁇ L of each tissue homogenate was added to 200 ⁇ L of acetonitrile, vortexed for 5 min, centrifuged at 10000 ⁇ g for 10 min, and the supernatant was taken for HPLC injection. The content of PTX in the tissue samples was calculated according to the linear equation.
  • the homogenized material did not interfere with the separation of PTX, and the retention time of PTX was about 8.1 min.
  • the statistical method of Kinetic 4.0 pharmacokinetic program was used to calculate the parameters of AUC 0- ⁇ and AUC 0-120h in the tissue after intravenous injection of Taxol and neutrophil delivery system with different surface potential drug-loaded liposomes.
  • Relative ratio (Re) brain targeting efficiency (Te).
  • Relative ratio refers to neutrophil delivery of different surface potential-loaded liposomes The ratio of the system to the AUC in the brain, the greater the relative uptake rate, indicating that the formulation is more targeted to the brain tissue than the Taxol.
  • Brain targeting efficiency refers to the ratio of AUC in the brain to the AUC in other tissues. Te greater than 1 indicates that the selectivity of the preparation for the brain is greater than that of other tissues, Te is larger. This indicates that the formulation is more selective for the brain relative to the other tissues being compared.
  • the neutrophil delivery systems loaded with different surface potential-loaded liposomes have brain Re (0-t) and Re (0-Int) compared to Taxol as shown in Figure 5, and their Te is greater than 1 ( Figure 6) shows that the neutrophil delivery system has good brain targeting and can quickly cross the blood-brain barrier (BBB) to effectively deliver drugs to brain tissue.
  • BBB blood-brain barrier
  • mice with postoperative glioma model were randomly divided into 5 groups, 12 in each group, which were postoperative saline group, commercially available formulation, and neutrophils loaded with different surface potential drug-loaded liposomes.
  • Cell delivery system group The dosage of the Taxol group was 5 mg PTX/kg, and the neutrophil delivery system with different surface potential loading liposomes was injected intravenously with about 5 ⁇ 10 6 NEs per dose. 5mg PTX/kg. All animals were dosed 1, 2, 3, 4, 6, 8, and 10, respectively, for a total of 7 doses.
  • the in situ vaccination of mouse glioma G422 was recorded as day 0, and the survival time of each group of mice was recorded, and the survival curve was drawn. The results are shown in Fig. 7.
  • Example 2 Targeting and pharmacodynamic evaluation of a levofloxacin cationic liposome neutrophil delivery system in a mouse model of Streptococcus pneumoniae
  • Example 2 The extraction and purification of neutrophils were the same as in Example 1, and 10 mg of levofloxacin was taken to prepare a levofloxacin cationic liposome. The same procedure as in Example 1 was carried out.
  • the drug loading and encapsulation efficiency were determined by HPLC method, and the particle diameter and potential were measured using a laser particle size analyzer as shown in Table 6.
  • Mouse model of pneumococcal pneumonia Take a male mouse weighing 18-22 g of clean grade, anesthetize with 10% chloral hydrate 0.1 ml/100 g (300 mg/kg), and inject 40 ⁇ L of prepared 10 6 CFU/ from the nasal cavity. mL S. pneumoniae suspension.
  • mice 72 inflammatory mice were randomly divided into 2 groups, 36 in each group. The rats were fasted overnight before administration. The two groups of tumor-bearing mice were injected intravenously with levofloxacin and levofloxacin cationic liposome neutrophils. The drug delivery system was dosed with levofloxacin 10 mg/kg, and the mice were sacrificed at 0.167h, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h and 120h after administration. Five tissues of heart, liver, spleen, kidney and lung were washed with physiological saline, and the residual physiological saline was blotted with a filter paper and weighed. Each tissue was treated as follows, and the concentration of levofloxacin contained in each tissue sample at each time point was measured by an HPLC method.
  • tissue sample (heart, liver, spleen, kidney, lung) of the inflammatory mouse was weighed and placed in a blood collection tube. 2 mL of physiological saline was added and dispersed into a tissue homogenate by a tissue homogenizer at a high speed. 200 ⁇ L of each tissue homogenate was added to 200 ⁇ L of acetonitrile, vortexed for 5 min, centrifuged at 10000 ⁇ g for 10 min, and the supernatant was taken for HPLC injection. The content of levofloxacin in the tissue sample was calculated according to the linear equation.
  • the homogenate of the tissue did not interfere with the separation of levofloxacin, and the retention time of levofloxacin was about 5.6 min.
  • the commercially available levofloxacin injection has the most liver distribution in the inflammatory model mice, followed by the heart, spleen, and kidney, and the inflammation site is less distributed; compared with the commercially available levofloxacin injection, the neutrophil delivery
  • the organs most distributed in the drug system are the lungs and spleen, followed by the liver organs, and less distributed in other organs.
  • the Kinetic 4.0 pharmacokinetic program statistical method was used to calculate the parameters such as AUC 0- ⁇ and AUC 0-120h in the tissue after intravenous injection of levofloxacin and neutrophil delivery system, and the relative ratio (relative ratio, Re was calculated). ), targeting efficiency (Te).
  • the relative ratio (Re) refers to the ratio of the neutrophil delivery system to the commercially available preparation levofloxacin in the lung AUC.
  • Targeting Brain targeting efficiency (Te) refers to the ratio of AUC in the lung to the AUC in other tissues. Te greater than 1 indicates that the selectivity of the preparation for the lung is greater than that of other tissues. The larger Te, the greater The more selective the formulation is to bacterially infective lung tissue relative to the other tissues being compared.
  • the inflammatory sites Re (0-t) and Re (0-Int) of the neutrophil delivery system compared to the commercially available levofloxacin are shown in Figure 9, and their Te is greater than 1 ( Figure 10), indicating neutral
  • the granulocyte delivery system has excellent inflammatory targeting and is capable of rapidly passing through inflammatory blood vessels and effectively delivering drugs to inflammatory tissues.
  • mice 48 inflammatory model mice were randomly divided into 4 groups, 12 rats in each group, which were postoperative saline group, simple neutrophil group, commercial preparation group and neutrophil delivery system group. The group was administered at a dose of 10 mg/kg, and the neutrophil delivery system group was intravenously injected with about 5 x 10 6 NEs per dose at a dose of about 10 mg/kg. All animals were dosed 1, 2, 3, 4, 6, 8, and 10, respectively, for a total of 7 doses. At the same time, the same cell density was given, and the same physiological saline volume group was used as a blank control. The result is shown in FIG.
  • the commercial preparation of levofloxacin had the same survival time as the normal saline in the model mice, and the neutrophil delivery system effectively prolonged the survival of the mice at 42 days, and the mice survived. It is proved that the neutrophil delivery system prepared by the present invention effectively inhibits the occurrence and development of infectious inflammation.
  • Example 1 The extraction and purification of neutrophils were the same as in Example 1, and 5 mg of ibuprofen was taken to prepare a load ibuprofen cationic liposome as in Example 1.
  • the drug loading and the encapsulation efficiency were measured by HPLC, and the particle diameter and potential were measured using a laser particle size analyzer as shown in Table 9.
  • Mouse model of ear swelling Take a male mouse with a body weight of 18-22 g, and a circular filter paper with a diameter of 7 mm saturated with xylene was attached to the right ear of the mouse for 15 s.
  • mice 72 inflammatory mice were randomly divided into 2 groups, 36 in each group. The rats were fasted overnight before administration. The two groups of tumor-bearing mice were intravenously injected with commercial ibuprofen and ibuprofen cationic liposome. The neutrophil delivery system was given ibuprofen 15 mg/kg at 0.167h, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h and 120h after administration. After taking the eyeball to take blood, the mice were sacrificed, and five tissues of heart, liver, spleen, kidney and ear were taken, washed with physiological saline, and the residual physiological saline was blotted with a filter paper and weighed. Each tissue was treated as follows, and the concentration of ibuprofen contained in each tissue sample at each time point was measured by an HPLC method.
  • Tissue samples (heart, liver, spleen, kidney, ear) of the inflamed mice were weighed and placed in a blood collection tube. 2 mL of physiological saline was added and dispersed into a tissue homogenate by a tissue homogenizer at high speed. 200 ⁇ L of each tissue homogenate was added to 200 ⁇ L of acetonitrile, vortexed for 5 min, centrifuged at 10000 ⁇ g for 10 min, and the supernatant was taken for HPLC injection. The content of PTX in the tissue samples was calculated according to the linear equation.
  • the homogenate of the tissue did not interfere with the separation of ibuprofen, and the retention time of ibuprofen was about 10.2 min.
  • the commercially available ibuprofen injection has the most liver distribution in the inflammatory model mice, followed by heart, lung, spleen and kidney, and the inflammation site is less distributed; compared with the commercially available ibuprofen injection,
  • the organs most distributed by the neutrophil delivery system are the ear and spleen of the inflammation site, followed by organs such as the liver and lungs, and are less distributed in other organs.
  • the Kinetic 4.0 pharmacokinetic program statistical method was used to calculate the parameters of AUC 0- ⁇ and AUC 0-120h in the tissues after intravenous ibuprofen and neutrophil delivery system, and the relative ratio (Re) was calculated. Brain targeting efficiency (Te).
  • the relative ratio (Re) refers to the ratio of the neutrophil delivery system to the commercially available preparation ibuprofen in the ear, and the higher the relative uptake rate, indicating that the preparation is relative to ibuprofen in the inflammatory tissue ear. The more targeted, the more targeted.
  • Targeting effect Brain targeting efficiency (Te) refers to the ratio of AUC in the ear to the AUC in other tissues. Te greater than 1 indicates that the selectivity of the preparation for the brain is greater than that of other tissues compared. The larger Te, the greater The more selective the formulation is to the ear of non-specific inflammation sites relative to the other tissues being compared.
  • the inflammatory sites Re (0-t) and Re (0-Int) of the neutrophil delivery system compared with the commercially available preparation ibuprofen are shown in Figure 13, and their Te is greater than 1 (Fig. 14), indicating The neutrophil delivery system has excellent inflammatory targeting and is capable of rapidly passing through inflammatory blood vessels and effectively delivering drugs to inflammatory tissues.
  • mice 48 inflammatory model mice were randomly divided into 4 groups, 12 rats in each group, which were postoperative saline group, simple neutrophil group, commercial preparation group and neutrophil delivery system group. Twelve tumor mice were used as a control group. Among them, the commercially available preparation group was administered at a dose of 15 mg/kg, and the neutrophil delivery system group was intravenously injected with about 5 x 10 6 NEs per dose at a dose of about 15 mg/kg. All animals were dosed 1, 2, 3, 4, 6, 8, and 10, respectively, for a total of 7 doses. At the same time, the same cell density was given, and the same physiological saline volume group was used as a blank control.
  • mice After continuous administration for 3 days, after 3 times of administration, the mice were sacrificed by cervical dislocation, and the discs of the same part of the left ear and the right ear were washed with a 7 mm puncher and weighed on the analytical balance, respectively, and the difference between the weights of the two ears was expressed.
  • the degree of swelling compares the anti-inflammatory effects of the drug, as shown in Figure 15. Compared with the saline group, the commercial preparation of ibuprofen and simple neutrophils did not significantly improve the degree of ear swelling, but the degree of ear swelling was significantly improved compared with the drug-loaded neutrophil group, t test. Found p ⁇ 0.05. It was demonstrated that the neutrophil delivery system prepared by the present invention effectively inhibits the occurrence and development of non-specific inflammation.
  • Example 1 The extraction and purification of neutrophils were the same as in Example 1, and 10 mg of ibuprofen was prepared.
  • the method for preparing the ibuprofen cationic liposome was the same as in Example 1.
  • the drug loading and the encapsulation efficiency were measured by HPLC, and the particle diameter and potential were measured using a laser particle size analyzer as shown in Table 12.
  • mice 72 inflammatory mice were randomly divided into 2 groups, 36 in each group. The rats were fasted overnight before administration. The two groups of tumor-bearing mice were injected intravenously with levofloxacin and ibuprofen cationic liposome. The granulocyte delivery system was administered with ibuprofen 15 mg/kg at 0.167h, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h and 120h, respectively. After taking blood from the eyeball, the mice were sacrificed, and five tissues of heart, liver, spleen, kidney and right hind paw were taken, washed with physiological saline, and dried with filter paper to dry the residual physiological saline. Each tissue was treated as follows, and the concentration of ibuprofen contained in each tissue sample at each time point was measured by an HPLC method.
  • Tissue samples of the tumor-bearing mice were weighed and placed in blood collection tubes. 2 mL of physiological saline was added and dispersed into a tissue homogenate by a tissue homogenizer at high speed. 200 ⁇ L of each tissue homogenate was added to 200 ⁇ L of acetonitrile, vortexed for 5 min, centrifuged at 10000 ⁇ g for 10 min, and the supernatant was taken for HPLC injection. The content of ibuprofen in the tissue sample was calculated according to the linear equation.
  • the homogenate of the tissue did not interfere with the separation of ibuprofen, and the retention time of ibuprofen was about 10.2 min.
  • the commercially available ibuprofen injection has the most liver distribution in the inflammatory model mice, followed by the heart, spleen and kidney, and the inflammation site is less distributed; compared with the commercially available ibuprofen injection, the neutral capsule
  • the organs most distributed in the cell delivery system are the paws and spleen of the inflammation site, followed by the liver organs, and less distributed in other organs.
  • the Kinetic 4.0 pharmacokinetic program statistical method was used to calculate the parameters of AUC 0- ⁇ and AUC 0-120h in the tissues after intravenous ibuprofen and neutrophil delivery system, and the relative ratio (Re) was calculated. Brain targeting efficiency (Te).
  • the relative ratio (Re) refers to the ratio of the neutrophil delivery system to the commercially available preparation ibuprofen in the paw AUC.
  • Brain targeting efficiency (Te) refers to the same formulation in the AUC of the paws of the inflammation site and other tissues.
  • the ratio of AUC, Te greater than 1 indicates that the selectivity of the preparation for the right hind paw of the inflammation site is greater than that of the other tissues compared, and the larger Te, the lower right of the preparation for the non-specific inflammation site relative to the other tissues being compared. The selectivity of the ankle is stronger.
  • the inflammatory sites Re (0-t) and Re (0-Int) of the neutrophil delivery system compared with the commercial preparation of ibuprofen are shown in Figure 17, and their Te is greater than 1 ( Figure 18), indicating The neutrophil delivery system has excellent inflammatory targeting and is capable of rapidly passing through inflammatory blood vessels and effectively delivering drugs to inflammatory tissues.
  • mice 48 inflammatory model mice were randomly divided into 4 groups, 12 in each group, respectively.
  • the inflammatory model was established after the saline group, the simple neutrophil group, the commercial preparation group, and the neutrophil delivery system group.
  • the drug-administered group was administered at a dose of 15 mg/kg
  • the neutrophil-delivery system group was intravenously injected with about 5 x 10 6 NEs per dose at a dose of about 15 mg/kg. All animals were dosed 1, 2, 3, 4, 6, 8, and 10, respectively, for a total of 7 doses.
  • the same cell density was given, and the same physiological saline volume group was used as a blank control.
  • the arthritis index score was performed every 3 days, and secondary lesions of each group of rats were observed.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Botany (AREA)
  • Dispersion Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Toxicology (AREA)
  • Urology & Nephrology (AREA)
  • Endocrinology (AREA)
  • Pathology (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Radiology & Medical Imaging (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)

Abstract

一种炎症靶向的中性粒细胞递药系统及其应用，所述的递药系统由中性粒细胞以及以直接或间接地方式载到中性粒细胞内或表面的治疗性物质或者检测性物质组成。将中性粒细胞作为药物的载体，能够将药物主动靶向到炎症部位，提高药物在炎症部位的浓度。到达炎症部位的中性粒细胞在细胞因子的刺激下，异常激活，快速解体以"天网"形式死亡，有利于将所荷载的药物快速释放到靶部位，提高治疗效果，降低毒副作用。

Description

一种炎症靶向的中性粒细胞递药系统及其应用 技术领域
本发明属于药物制剂领域，涉及一种炎症靶向的中性粒细胞递药系统及其应用，具体涉及一种以中性粒细胞为载体的靶向炎症部位的递送系统。
背景技术
炎症指生物组织受到外伤、出血、或病原感染等刺激，激发的生理反应，其中包括红肿、发热、疼痛等症状。炎症反应是先天免疫系统为移除有害刺激或病原体及促进修复的一种重要防御机制，根据发病的原因分为感染性炎症、非特异性炎症及变态反应性炎症。感染性炎症是由病毒、细菌或细菌产物造成的炎症，通常用抗生素或抗真菌药物来治疗；非特异性炎症是用某种致炎因子于动物体内或体外引起炎症或其某个阶段的细胞组织反应，如缺血再灌注性损伤、术后损伤等，需要系统性治疗；变态反应性炎症是多型变态反应的终末表现如狼疮性皮炎，需要抗免疫治疗。
目前，针对炎症组织全身给药后，由于其独特的病理特征，使得药物的靶向效率较低，全身毒副作用较大。炎症的病理特征主要包括局部组织变质、渗出和增生。由于炎症血管具有较高的通透性，血管内富含蛋白质的液体和白细胞渗出，导致炎症局部组织间质压升高。可见，炎症部位血管的高渗透性为药物从血管溢出并滞留于炎症部位提供了可能，但是由于较高的炎症组织间质压阻碍了药物进入炎症部位。由于抗炎药物较低的靶向性，使得一些感染性炎症如细菌性肺炎，治疗不及时，从而产生耐药性导致较高的死亡率。而对于一些炎症相关疾病的治疗，如缺血再灌注性脑损伤、术后肿瘤复发等，由于原发灶在周围被非特异性炎症组织所包围，治疗药物靶向效率降低，药效下降，副作用增加。
如何以最小的剂量达到最佳的诊断和治疗效果一直以来都是困扰药剂工作者们的关键性问题，随着纳米技术领域的发展和人们对生物体的不断深入了解，靶向递送系统应运而生。靶向递送系统主要分为三大类：被动靶向、主动靶向以及物理化学靶向。与以往的传递系统相比，虽然这些新兴的靶向递送系统已经有了质的飞跃，但是仍然存在靶向性差，毒副作用强，诊断治疗效果难以达到人们预期等的缺点，因此，极其需要寻找一种更为高效、低毒的靶向递送系统。
免疫细胞，包括单核吞噬细胞(树状突细胞，单核细胞和巨噬细胞)，中性粒细胞和淋巴 细胞。炎症过程中免疫细胞的渗出是炎症反应最为重要的特征，而中性粒细胞作为炎症发生时的“防御卫士”，被趋化因子吸引能够第一时间到达炎症部位。目前，仍未见中性粒细胞作为载体用于炎症部位药物、探针和显影剂递送研究的相同或相近报导。药物或纳米制剂荷载到中性粒细胞内存在一些有待解决的科学问题，如药物荷载的方式，细胞荷载药物、探针和显影剂的量，所荷载药物在细胞内稳定性以及细胞载体固有生理活性的维持等问题。
发明内容
为了克服上述现有技术的不足，本发明提供了靶向炎症组织的荷载药物的中性粒细胞递药系统。
本发明的另一目的是提供该递药系统的应用。
本发明的又一目的是提供一种治疗炎症的药物。
一种炎症靶向的中性粒细胞递药系统，所述的递药系统由中性粒细胞以及以直接或间接地方式载到中性粒细胞内或表面的治疗性物质或者检测性物质组成。
其中，所述的治疗性物质包括但不限于药物(化学合成药物、天然药物或天然药物提取物)、具有治疗作用的DNA、RNA、蛋白质或多肽中的一种或多种；所述的检测性物质选自探针、显影剂中的一种或多种。
所述的间接地方式是指将纳米载体作为工具，将治疗性物质或者检测性物质先装载在纳米载体内，组成纳米制剂，再将纳米制剂荷载到中性粒细胞内或表面。
所述的纳米载体优选自正电性、负电性或近中性的粒径为1-1000nm纳米制剂。
所述的纳米载体包括但不限于金纳米粒/棒、磁性纳米粒、介孔二氧化硅纳米粒、石墨烯、脂质体、胶束、纳米乳、纳米球、纳米囊、微球、微丸或树枝状聚合物。
所述的药物选自传出神经系统药物、中枢神经系统药、心血管系统药、激素及呼吸消化系统药、抗菌或抗病毒药物、抗肿瘤药物、维生素中的一种或多种。
所述的药物优选自解热镇痛类药物、非甾体抗炎药物、抗肿瘤类药物、抗生素类药物、利尿药、降压药、降脂药、降血糖药、激素类药物或维生素类药物中的一种或多种。
所述的药物进一步优选地西泮、苯妥英钠、氯丙嗪、氟西汀、美沙酮、甲氯芬酯、氯贝胆碱、硫酸阿托品、异丙肾上腺素、马来酸氯苯那敏、盐酸普鲁卡因、盐酸普萘洛尔、盐酸维拉帕米、盐酸胺碘酮、氯沙坦、硝酸甘油、多巴酚丁胺、辛伐他汀、氯吡格雷、哌唑嗪、西咪替丁、地芬尼多、西沙比利、联苯双酯、阿司匹林、吲哚美辛、盐酸氮芥、氟尿嘧啶、紫杉醇、 阿莫西林、四环素、氨基糖苷、罗红霉素、氯霉素、左氟沙星、异烟肼、磺胺嘧啶、氟康唑、盐酸金刚烷胺、磷酸氟喹、格列苯脲、氢氯噻嗪、前列腺素、胰岛素、雌二醇、维生素A、维生素C中的一种或多种。
本发明所述的炎症靶向的中性粒细胞递药系统在制备治疗或诊断炎症和/或炎症相关疾病的药物或试剂中的应用。
其中，所述的炎症包括感染性炎症、非特异性炎症、变态反应性炎症以及炎症相关性疾病。
所述的感染性炎症包括病毒、细菌或细菌产物造成的炎症；所述的非特异性炎症包括即物理性炎症，包括手术或外伤引起的红肿、疼痛等；变态反应性炎症包括狼疮性皮炎、过敏性哮喘、风湿性关节炎；炎症相关性疾病包括肿瘤术后复发的治疗、动脉粥样硬化、缺血缺氧性脑病。
本发明将治疗性物质或者检测性物质先包覆在不同电性的纳米载体中(正电性、负电性、近中性，1-1000nm)，再将载药纳米载体(即纳米制剂)荷载到中性粒细胞内/表面；或者直接将治疗性物质或者检测性物质荷载到中性粒细胞内/表面，成功制备了以中性粒细胞为载体的递送系统。将所制备的中性粒细胞递药系统静脉注射到人体内，血液循环过程中，受到炎症区域释放的趋化因子的刺激，穿过炎症部位血管，沿着趋化因子浓度梯度，主动定位到炎症部位，提高了炎症部位药物浓度，保障了药效的发挥。研究表明，感染性炎症、非特异性炎症、变态反应性炎症以及炎症相关性疾病如肿瘤手术所引起的急性炎症都与中性粒细胞募集有关，因此利用中性粒细胞作为递药系统治疗炎症或炎症相关的疾病如肿瘤手术后利用急性炎症治疗肿瘤的复发，可以有效提高药物在靶部位的蓄积，提高药物的药效，降低其毒副作用。综上所述，荷载治疗性物质或者检测性物质的中性粒细胞，在炎症因子的引诱下，可以将治疗性物质或者检测性物质成功递送到炎症相关疾病部位，进行疾病的治疗和诊断，抑制疾病的发生和发展。
一种治疗炎症和/或炎症相关疾病的药物，包含本发明所述的炎症靶向的中性粒细胞递药系统。
所述的中性粒细胞递药系统中的治疗性物质可以为常规给药途径中对炎症和/或炎症相关疾病有治疗作用的药物，包括但不限于神经系统类药物、心脑血管类药、血液、呼吸、消化系统药、内分泌系统以及免疫调节药物如免疫增强药干扰素、免疫抑制剂环孢素等。
一种诊断炎症和/或炎症相关疾病的试剂，包含本发明所述的炎症靶向的中性粒细胞递药系统。
所述的中性粒细胞递药系统中的诊断性物质包括但不限于探针如DNA探针、荧光探针，造影剂如X线观察常用的碘制剂、硫酸钡以及核磁成像造影剂如钆喷酸葡胺等。
本发明中所涉及的某些概念定义如下：
纳米载体：即纳米级药物载体，是一种属于纳米级微观范畴的亚微粒药物载体输送系统。将药物包封于亚微粒(即纳米载体)中，可以调节释药的速度，增加生物膜的透过性、改变在体内的分布、提高生物利用度等。
本发明所述的纳米制剂可按照现有技术中报道过的任何一种方法制备。
脂质体制备：
(1)药物分散于有机相的脂质体制备方法
适合制备脂溶性药物的脂质体，具体说来这一类方法包括薄膜分散法、注入法、前体脂质体法、超声分散法等方法。
(2)药物分散于水相的脂质体制备方法
适合制备水溶性药物的脂质体，但要求药物有比较好的稳定性，具体说来这一类方法包括反相蒸发法、复乳法、熔融法、冻融法、冷冻干燥法、表面活性剂处理法、钙融合法、离心法等方法。
炎症：具有血管系统的活体组织对损伤因子所发生的防御反应为炎症。
有益效果：
中性粒细胞是数量最多的白细胞，占白细胞的50％～70％，容易从患者体内获得，这为药物递送提供了丰富的后备军。
本发明以中性粒细胞为递药载体，首先，中性粒细胞保护所载药物、探针或显影剂等免受网状内皮系统清除，延长半衰期，有效控制释放，降低免疫原性和毒副作用。其次，中性粒细胞作为载体可以穿过疏水屏障顺利到达发病区域，提高靶部位的浓度。第三，由于中性粒细胞具有趋化性质，能够靶向到损伤、炎症和肿瘤部位释药。因此，中性粒细胞作为低毒的转运体，可以起到靶向递送、延长循环时间，降低细胞和组织的毒性的作用。
所以，鉴于中性粒细胞作为递药系统具有上述优点，将中性粒细胞作为药物的载体，能够 将药物主动靶向到炎症部位，提高药物在炎症部位的浓度。到达炎症部位的中性粒细胞在细胞因子的刺激下，异常激活，快速解体以“天网”形式死亡，有利于将所荷载的药物快速释放到靶部位，提高治疗效果，降低毒副作用。
与现有技术相比，本发明具有的优点如下：
1.本发明成功制备的中性粒细胞递药系统，顺趋化因子浓度梯度到达炎症组织，成功解决了药物、探针或显影剂等的靶向性问题。
2.本发明所制备的中性粒细胞递药系统，通过纳米载体的介导，保证了中性粒细胞较高的载药量，并且避免了药物对中性粒细胞生理活性的影响。
3.本发明所制备的中性粒细胞递送系统，由于中性粒细胞是机体的一种天然免疫细胞，吞噬能力强，因此细胞所载药物、探针或显影剂的量很高。
4.本发明所使用的药物载体中性粒细胞，是人体内数量最多的免疫细胞，容易从患者体内获得，并且中性粒细胞在炎症部位异常激活后，会以独特的死亡方式裂解，能够将所荷载的药物快速、彻底的释放到炎症组织，这使得本发明具备较高的临床开发应用价值。
附图说明
图1瑞氏-吉姆萨对65％/78％Percoll分离层形态学鉴定。
图2不同表面电位载药脂质体的释放。
图3中性粒细胞对不同电性脂质体的摄取量。
图4三种电性紫杉醇脂质体在体外模拟不同生理病理条件下在中性粒细胞中的滞留。
图5术后脑胶质瘤模型小鼠给药后炎症部位荷载负电性、近中性、正电性纳米制剂的中性粒细胞递药系统相对于市售制剂的相对摄取率。
图6小鼠给药后市售制剂与荷载负电性、近中性、正电性纳米制剂的中性粒细胞递药系统靶向效率。
图7脑胶质瘤术后模型小鼠给药生理盐水、空白中性粒细胞、市售制剂泰素、荷载负电性、近中性、正电性纳米制剂的中性粒细胞递药系统生存期曲线。
图8三种电性左氧氟沙星脂质体在体外模拟不同生理条件下在中性粒细胞中的滞留
图9肺炎链球菌肺炎模型小鼠给药后炎症部位中性粒细胞递药系统相对于市售制剂的相对摄取率。
图10小鼠给药后市售制剂与中性粒细胞递药系统靶向效率。
图11肺炎链球菌肺炎模型小鼠给药生理盐水、空白中性粒细胞、市售制剂左氧氟沙星、载药中性粒细胞递药系统生存期曲线。
图12三种电性布洛芬脂质体在体外模拟不同生理条件下在中性粒细胞中的滞留
图13耳肿胀模型小鼠给药后炎症部位中性粒细胞递药系统相对于市售制剂的相对摄取率。
图14小鼠给药后市售制剂与中性粒细胞递药系统靶向效率。
图15耳肿胀模型小鼠小鼠给药生理盐水、空白中性粒细胞、市售制剂布洛芬、载药中性粒细胞递药系统耳肿胀程度。
图16三种电性布洛芬脂质体在体外模拟不同生理条件下在中性粒细胞中的滞留
图17佐剂性关节炎模型小鼠给药后炎症部位中性粒细胞递药系统相对于市售制剂的相对摄取率。
图18小鼠给药后市售制剂与中性粒细胞递药系统靶向效率。
图19佐剂性关节炎模型小鼠给药生理盐水、空白中性粒细胞、市售制剂布洛芬、载药中性粒细胞递药系统关节炎指数评分。
具体实施方式
以下结合各实施例对本发明的技术方案做进一步说明，实施例仅是举例说明本发明的技术方案及其效果，并不应作为对本发明保护范围的限制。
实施例1荷载不同表面电位紫杉醇脂质体中性粒细胞递药系统对术后脑胶质瘤小鼠模型的靶向性及药效学评价
一、骨髓中性粒细胞的分离和纯化
将Percoll母液与10×PBS按9:1(v/v)制备成100％Percoll，以1×PBS稀释制备成55％、65％、68％(v/v)Percoll分离液。按照分离液密度先重后轻的顺序，缓慢均匀叠加制备55％、65％的两层Percoll分离液，将新鲜提取的小鼠胫骨骨髓细胞用1×PBS稀释制备成单细胞悬液，1:1(v/v)缓慢均匀加在之前制备55％、65％Percoll分离液顶部，1000g离心30min。提取55％、65％分离液夹层的骨髓细胞，加入一倍体积1×PBS洗涤并重悬，再缓慢叠加到68％的Percoll分离液上1000g离心30min，提取68％分离液层的骨髓细胞，加入一倍体积1×PBS洗涤，重复两次，RPMI1640培养基重悬制备小鼠骨髓中性粒细胞悬液，备用。
用红色荧光标记的细胞表面蛋白Gr-1抗体(北京达科为生物科技有限公司)对提取细胞进行染色；37℃、5％CO2孵育30min，流式细胞仪检验所提取细胞纯度。用台盼蓝对提取细胞进行活力检测细胞活力为98％左右；用瑞氏-吉姆萨染色液对提取细胞进行形态学纯度鉴定，结果如图1所示，分离得到的中性粒细胞细胞核大多呈成熟杆状或分叶状；用红色荧光标记的细胞表面蛋白Gr-1抗体对提取细胞进行染色，结果发现所提取中性粒细胞纯度高于90％。
二、紫杉醇-脂质体-中性粒细胞(PTX-Liposome-NEs)递药系统的制备
1.荷载紫杉醇的不同电性脂质体的制备
利用薄膜分散法制备荷载紫杉醇的三种不同电性的脂质体，制备步骤具体为：
精密称取处方量的下列各物质
1)正电性脂质体：大豆磷脂90mg+阳离子脂质10mg+胆固醇10mg+紫杉醇5mg；
2)近中性脂质体：中性脂质100mg+胆固醇10mg+紫杉醇5mg；
3)负电性脂质体：大豆磷脂100mg+胆固醇10mg+紫杉醇5mg；
将处方量各物质溶于氯仿/甲醇(2:1,v:v)混合溶剂中，混匀后，40℃水浴抽真空旋干成膜，并在真空干燥器中放置过夜以完全除去有机溶剂后，用超纯水37℃下水化脂膜，得到的脂质混悬液用超声波细胞粉粹机冰浴分散，分别过0.45μm和0.22μm微孔滤膜，制得三种不同电性的荷载紫杉醇的脂质体(PTX-Liposome)。
使用HPLC法测定PTX-Liposome的载药量和包封率，使用激光粒径分析仪测定粒径和电位，如表1所示。
表1
Figure PCTCN2014092340-appb-000001
采用透析法测定体外释放，结果见图2。结果表明，所得脂质体在测定时间范围内释放缓慢，24h累计释放量不足20％，具有一定的缓释特性。
2.紫杉醇-脂质体-中性粒细胞递药系统(PTX-Liposome-NEs)的制备
取新鲜制备的中性粒细胞悬液以1×106个/孔、500μL接种于24孔板中，不含血清RPIM1640培养液37℃、5％CO2稳定培养1h，移去培养液，将三种不同电性的紫杉醇脂质体用不含血清RPIM1640培养液稀释成PTX 100μg/ml加入上述24孔板，于37℃孵育50min后， 弃去含制剂的培养液，用4℃PBS洗三次，即得不同表面电位的紫杉醇脂质体-中性细胞递药系统。
使用HPLC测定中性粒细胞对不同电性脂质体的摄取量，结果见图3。结果表明，中性粒细胞对正电性脂质体的摄取量明显高于近中性和负电性的脂质体，为制备载药量较高的递药系统提供了保证。
体外利用Ph7.4RPMI1640培养基、10nM趋化三肽以及100nM佛波醇分别模拟正常生理条件、趋化过程以及炎症部位三种生理病理环境，使用HPLC测定了4h三种电性紫杉醇脂质体在不同生理病理条件下在中性粒细胞中的滞留，结果见图4。结果表明，细胞递药系统在趋化过程中药物累计释放量仅占总摄取量的5％，释放缓慢，而到达靶部位，中性粒细胞异常激活情况下，药物累计释放量占总摄取量的85％，保证了大部分药物能够到达靶部位并发挥疗效。
三、术后脑胶质瘤模型小鼠建立
将KM小鼠用水合氯醛腹腔注射麻醉后固定于立体定向头架，将小鼠脑胶质瘤细胞G422以微量注射器注射于小鼠右侧尾状核(手术暴露颅骨标志，以牙钻于前囟右旁开4mm，前1mm，孔径1.2mm，硬膜下进针深5mm)。缝合，消毒后放回笼内。
四、紫杉醇-脂质体-中性粒细胞递药系统(PTX-Liposome-NEs)的术后脑胶质瘤模型小鼠的相对摄取率和靶向效率。
1.给药方案
术后脑胶质瘤模型小鼠144只，随机分为4组，每组36只，给药前禁食不禁水过夜，4组荷瘤小鼠尾分别静脉注射紫杉醇市售制剂泰素(Taxol)和荷载不同表面电位载药脂质体的中性粒细胞递药系统，PTX剂量均为5mg/kg，分别于给药后0.167h、0.5h、1h、2h、4h、8h、12h、24h、48h、72h、96h和120h时间点，每组取3只荷瘤小鼠，摘眼球取血后，处死小鼠，取心、肝、脾、肾、脑5个组织，用生理盐水洗净，并用滤纸吸干残留生理盐水后称重。各组织按下述方法处理，用HPLC方法测各时间点的各组织样品中所含PTX浓度。
2.样品处理
取荷瘤小鼠各组织样品(心、肝、脾、肺、肾、脑)，称重，置于采血管中，加入2mL的生理盐水，经组织匀浆器高速分散成组织匀浆液。取200μL各组织匀浆液加入200μL乙腈，涡旋5min后，10000×g离心10min，取上清液，HPLC进样，根据线性方程计算组织样品中PTX的含量。
3.HPLC方法
色谱柱：
Figure PCTCN2014092340-appb-000002
ODS-SP column(250mm×4.6mm×5μm,GL Sciences Inc.,Japan)
流动相：甲醇:水＝80:20(v:v)
检测波长：227nm
柱温：35℃
流速：1.0mL/min
进样量：20μL
组织匀浆的物质不干扰PTX的分离测定，PTX的保留时间约为8.1min。
5.小鼠体内分布结果
表2泰素(Taxol)在各脏器中分布
Figure PCTCN2014092340-appb-000003
表3荷载正电性纳米制剂中性粒细胞递药系统在各脏器的分布
Figure PCTCN2014092340-appb-000004
表4荷载近中性纳米制剂中性粒细胞递药系统在各脏器的分布
Figure PCTCN2014092340-appb-000005
表5荷载负电性纳米制剂中性粒细胞递药系统在各脏器的分布
Figure PCTCN2014092340-appb-000006
HPLC结果显示：市售制剂泰素在肝脏分布最多，其次为心、脾脏、肾脏，在脑中分布极少；与泰素相比，中性粒细胞递药系统组分布最多的器官为脑和脾脏，其次为肝脏和肾等器官，在其他器官分布较少；荷载不同电位纳米制剂中性粒细胞递药系统各组比较，各脏器中分布没有显著性差异。
5.靶向性评价
应用Kinetic 4.0药代动力学程序统计方法计算静脉注射泰素和荷载不同表面电位载药脂质体的中性粒细胞递药系统后组织中AUC0-∞、AUC0-120h等参数，计算相对摄取率(relative ratio,Re)、靶向效率(brain targeting efficiency,Te)。
相对摄取率(relative ratio,Re)是指荷载不同表面电位载药脂质体的中性粒细胞递药 系统与泰素在脑中AUC的比值，相对摄取率越大，表明该制剂相对于泰素在脑组织的靶向性越强。靶向效率(brain targeting efficiency,Te)是指同一种制剂在脑中的AUC与其他组织中AUC的比值，Te大于1表示该制剂对于脑的选择性大于相比较的其他组织，Te越大，则表明相对于被比较的其他组织，该制剂对脑的选择性越强。
荷载不同表面电位载药脂质体的中性粒细胞递药系统与泰素相比的脑Re(0-t)和Re(0-Int)如图5所示，并且其Te均大于1(图6)，说明中性粒细胞递药系统具有很好的脑靶向性，能够快速的穿过血脑屏障(Brain-Blood-Barrier,BBB)，将药物有效传递到脑组织。
五、载药中性粒细胞递药系统对术后脑胶质瘤模型小鼠药效学评价
术后脑胶质瘤模型小鼠60只，随机分成5组，每组12只，分别为术后生理盐水组、市售制剂泰素组、荷载不同表面电位载药脂质体的中性粒细胞递药系统组。其中泰素组的给药剂量为5mg PTX/kg，荷载不同表面电位载药脂质体的中性粒细胞递药系统组每只静脉注射约5×106个NEs/只，给药剂量约5mg PTX/kg。所有动物分别在术后1、2、3、4、6、8、10给药，共给药7次。以原位接种小鼠脑胶质瘤G422记为第0天，记录个组小鼠的生存时间，绘制生存期曲线，结果见图7。由图7可见，荷载不同表面电位载药脂质体的中性粒细胞递药系统组能够显著延长术后脑胶质瘤模型小鼠的生存时间。证明本发明所制备的中性粒细胞递药系统有效的抑制炎症相关性疾病的发生及发展。
实施例2荷载左氧氟沙星阳离子脂质体中性粒细胞递药系统对肺炎链球菌小鼠模型的靶向性及药效学评价
一、载药中性粒细胞递药系统的制备及表征
中性粒细胞的提取与纯化同实施例1，取10mg左氧氟沙星，制备荷载左氧氟沙星阳离子脂质体，方法同实施例1。
使用HPLC法测定载药量和包封率，使用激光粒径分析仪测定粒径和电位，如表6所示。
表6
Figure PCTCN2014092340-appb-000007
使用HPLC测定三种电性左氧氟沙星脂质体在体外模拟不同生理条件下在中性粒细胞中的滞留，结果见图8由图可见，在模拟正常血液循环以及炎症因子趋化过程中，药物的释放量仅占总摄取量的7％，但在炎症部位，在中性粒细胞异常激活情况下，药物释放量占总摄取量的 90％，证明在炎症部位药物可以快速而彻底的从细胞中释放出来。
二、炎症动物模型建立
肺炎链球菌肺炎小鼠模型：取体重18～22g清洁级雄性小鼠，以10％水合氯醛0.3ml/100g(300mg/kg)腹腔注射麻醉，从鼻腔滴入40μL制备好的106CFU/mL肺炎链球菌悬液。
三、中性粒细胞递药系统炎症模型小鼠靶向性研究
1.给药方案
取炎症小鼠72只，随机分为2组，每组36只，给药前禁食不禁水过夜，2组荷瘤小鼠尾分别静脉注射市售左氧氟沙星以及左氧氟沙星阳离子脂质体中性粒细胞递药系统，剂量均为左氧氟沙星10mg/kg，分别于给药后0.167h、0.5h、1h、2h、4h、8h、12h、24h、48h、72h、96h和120h时间点，处死小鼠，取心、肝、脾、肾、肺5个组织，用生理盐水洗净，并用滤纸吸干残留生理盐水后称重。各组织按下述方法处理，用HPLC方法测各时间点的各组织样品中所含左氧氟沙星浓度。
2.样品处理
炎症小鼠各组织样品(心、肝、脾、肾、肺)，称重，置于采血管中，加入2mL的生理盐水，经组织匀浆器高速分散成组织匀浆液。取200μL各组织匀浆液加入200μL乙腈，涡旋5min后，10000×g离心10min，取上清液，HPLC进样，根据线性方程计算组织样品中左氧氟沙星的含量。
3.HPLC方法
色谱柱：
Figure PCTCN2014092340-appb-000008
ODS-SP column(250mm×4.6mm×5μm,GL Sciences Inc.,Japan)
流动相：甲醇:水＝80:20(v:v)以0.01mol/L磷酸二氢钾溶液(pH3.0)：乙腈＝45：55(v:v)
甲醇:乙腈:(0.01mol/L)磷酸二氢钾:(0.5mol/L)溴化四丁基铵＝(10:10:80:4)磷酸调
pH3.5
检测波长：294nm
柱温：室温
流速：1.0mL/min
进样量：20μL
组织匀浆的物质不干扰左氧氟沙星的分离测定，左氧氟沙星的保留时间约为5.6min。
6.小鼠体内分布结果
表7左氧氟沙星在各脏器中分布
Figure PCTCN2014092340-appb-000009
表8左氧氟沙星阳离子脂质体中性粒细胞递药系统在各脏器的分布
Figure PCTCN2014092340-appb-000010
由表7～8可知，市售左氧氟沙星注射剂在炎症模型小鼠体内情况，肝脏分布最多，其次为心、脾脏、肾脏，炎症部位分布较少；与市售左氧氟沙星注射剂相比，中性粒细胞递药系统分布最多的器官为肺和脾脏，其次为肝器官，在其他器官分布较少。
5.靶向性评价
应用Kinetic 4.0药代动力学程序统计方法计算静脉注射市售注射液左氧氟沙星、中性粒细胞递药系统后组织中AUC0-∞、AUC0-120h等参数，计算相对摄取率(relative ratio,Re)、靶向效率(brain targeting efficiency,Te)。
相对摄取率(relative ratio,Re)是指中性粒细胞递药系统与市售制剂左氧氟沙星在肺中AUC的比值，相对摄取率越大，表明该制剂相对于市售制剂在炎症组织的靶向性越强。靶向 效率(brain targeting efficiency,Te)是指同一种制剂在肺中的AUC与其他组织中AUC的比值，Te大于1表示该制剂对于肺的选择性大于相比较的其他组织，Te越大，则表明相对于被比较的其他组织，该制剂对细菌感染性肺组织的选择性越强。
中性粒细胞递药系统与市售制剂左氧氟沙星相比的炎症部位Re(0-t)和Re(0-Int)如图9所示，并且其Te均大于1(图10)，说明中性粒细胞递药系统具有很好的炎症靶向性，能够快速的穿过炎症血管，将药物有效传递到炎症组织。
四、中性粒细胞递药系统对肺炎链球菌肺炎小鼠模型药效学评价
炎症模型小鼠48只，随机分成4组，每组12只，分别为术后生理盐水组、单纯中性粒细胞组、市售制剂组、中性粒细胞递药系统组，其中市售制剂组的给药剂量为10mg/kg，中性粒细胞递药系统组每只静脉注射约5×106个NEs/只，给药剂量约10mg/kg。所有动物分别在术后1、2、3、4、6、8、10给药，共给药7次。同时设给予相同细胞密度，相同生理盐水体积组作为空白对照。结果如图11所示。市售制剂左氧氟沙星对模型小鼠的生存期与生理盐水相仿，都在21天左右，而中性粒细胞递药系统有效延长了小鼠的生存期在42天，仍有小鼠存活。证明本发明所制备的中性粒细胞递药系统有效的抑制感染性炎症的发生及发展。
实施3荷载布洛芬阳离子脂质体中性粒细胞递药系统对耳肿胀小鼠模型的靶向性及药效学评价
一、载药中性粒细胞递药系统的制备及表征
中性粒细胞的提取与纯化同实施例1，取5mg布洛芬，制备荷载布洛芬阳离子脂质体同实施例1。
使用HPLC法测定载药量和包封率，使用激光粒径分析仪测定粒径和电位，如表9所示。
表9
Figure PCTCN2014092340-appb-000011
使用HPLC测定三种电性布洛芬脂质体在体外模拟不同生理条件下在中性粒细胞中的滞留，结果见图12由图可见，在模拟正常血液循环以及炎症因子趋化过程中，细胞递药系统在趋化过程中药物累计释放量仅占总摄取量的7％，释放缓慢，但在炎症部位，中性粒细胞异常激活情况下，药物释放量占总摄取量的87％，证明在炎症部位药物可以快速而彻底的从细胞中释放出来。
二、炎症动物模型建立
耳肿胀小鼠模型：取体重18～22g清洁级雄性小鼠，将已浸透二甲苯的直径为7mm的圆形滤纸片紧贴在小鼠右耳部15s。
三、中性粒细胞递药系统炎症模型小鼠靶向性研究
1.给药方案
取炎症小鼠72只，随机分为2组，每组36只，给药前禁食不禁水过夜，2组荷瘤小鼠尾分别静脉注射市售布洛芬以及布洛芬阳离子脂质体中性粒细胞递药系统，剂量均为布洛芬15mg/kg，分别于给药后0.167h、0.5h、1h、2h、4h、8h、12h、24h、48h、72h、96h和120h时间点，摘眼球取血后，处死小鼠，取心、肝、脾、肾、耳5个组织，用生理盐水洗净，并用滤纸吸干残留生理盐水后称重。各组织按下述方法处理，用HPLC方法测各时间点的各组织样品中所含布洛芬浓度。
2.样品处理
取炎症小鼠各组织样品(心、肝、脾、肾、耳)，称重，置于采血管中，加入2mL的生理盐水，经组织匀浆器高速分散成组织匀浆液。取200μL各组织匀浆液加入200μL乙腈，涡旋5min后，10000×g离心10min，取上清液，HPLC进样，根据线性方程计算组织样品中PTX的含量。
3.HPLC方法
色谱柱：
Figure PCTCN2014092340-appb-000012
ODS-SP column(250mm×4.6mm×5μm,GL Sciences Inc.,Japan)
流动相：甲醇:水＝80:20(v:v)以0.01mol/L磷酸二氢钾溶液(pH3.0)：乙腈＝45：55(v:v)
检测波长：263nm
柱温：室温
流速：1.0mL/min
进样量：20μL
组织匀浆的物质不干扰布洛芬的分离测定，布洛芬的保留时间约为10.2min。
4.小鼠体内分布结果
表10布洛芬在各脏器中分布
Figure PCTCN2014092340-appb-000013
表11布洛芬阳离子脂质体中性粒细胞递药系统在各脏器的分布
Figure PCTCN2014092340-appb-000014
由上表可知，市售布洛芬注射剂在炎症模型小鼠体内情况，肝脏分布最多，其次为心、肺、脾脏、肾脏，炎症部位分布较少；与市售布洛芬注射剂相比，中性粒细胞递药系统分布最多的器官为炎症部位耳和脾脏，其次为肝脏和肺等器官，在其他器官分布较少。
5.靶向性评价
应用Kinetic 4.0药代动力学程序统计方法计算静脉注射布洛芬、中性粒细胞递药系统后组织中AUC0-∞、AUC0-120h等参数，计算相对摄取率(relative ratio,Re)、靶向效率(brain targeting efficiency,Te)。
相对摄取率(relative ratio,Re)是指中性粒细胞递药系统与市售制剂布洛芬在耳中AUC的比值，相对摄取率越大，表明该制剂相对于布洛芬在炎症组织耳的靶向性越强。靶向效 率(brain targeting efficiency,Te)是指同一种制剂在耳中的AUC与其他组织中AUC的比值，Te大于1表示该制剂对于脑的选择性大于相比较的其他组织，Te越大，则表明相对于被比较的其他组织，该制剂对非特异性炎症部位耳的选择性越强。
中性粒细胞递药系统与市售制剂布洛芬相比的炎症部位Re(0-t)和Re(0-Int)如图13所示，并且其Te均大于1(图14)，说明中性粒细胞递药系统具有很好的炎症靶向性，能够快速的穿过炎症血管，将药物有效传递到炎症组织。
四、中性粒细胞递药系统对耳肿胀小鼠模型药效学评价
炎症模型小鼠48只，随机分成4组，每组12只，分别为术后生理盐水组、单纯中性粒细胞组、市售制剂组、中性粒细胞递药系统组，同时设原位瘤小鼠12只，为对照组。其中市售制剂组的给药剂量为15mg/kg，中性粒细胞递药系统组每只静脉注射约5×106个NEs/只，给药剂量约15mg/kg。所有动物分别在术后1、2、3、4、6、8、10给药，共给药7次。同时设给予相同细胞密度，相同生理盐水体积组作为空白对照。连续给药3d，给药3次后，脱颈处死小鼠，用7mm打孔器冲下左耳和右耳同一部位的圆片于分析天平上分别称重，以两耳重量的差值表示肿胀程度，比较药物的抗炎作用，如图15所示。与生理盐水组相比，给予市售制剂布洛芬和单纯中性粒细胞对耳肿胀程度并没显著性改善，而与载药中性粒细胞组相比耳肿胀程度显著性改善，t检验发现p<0.05。证明本发明所制备的中性粒细胞递药系统有效的抑制非特异性炎症的发生及发展。
实施4荷载布洛芬阳离子脂质体中性粒细胞递药系统对佐剂性关节炎小鼠模型的靶向性及药效学评价
一、载药中性粒细胞递药系统的制备及表征
中性粒细胞的提取与纯化同实施例1，取10mg布洛芬，制备荷载布洛芬阳离子脂质体方法同实施例1。
使用HPLC法测定载药量和包封率，使用激光粒径分析仪测定粒径和电位，如表12所示。
表12
Figure PCTCN2014092340-appb-000015
使用HPLC测定三种电性布洛芬脂质体在体外模拟不同生理条件下在中性粒细胞中的滞留，结果见图16由图可见，在模拟正常血液循环以及炎症因子趋化过程中，药物的累计释放 量仅占总摄取量的8％，但在炎症部位，中性粒细胞异常激活情况下，药物累计释放量占总摄取量的91％，证明在炎症部位药物可以快速而彻底的从细胞中释放出来。
二、炎症动物模型建立
佐剂性关节炎小鼠模型：将卡介苗80℃水浴灭活1小时，与高压灭菌的石蜡充分研磨、混匀，制成10mg/ml完全弗氏佐剂(Complete Freund adjuvant，CFA)，于每只小鼠右后足跖皮内注射CFA 0.1ml致炎。
三、中性粒细胞递药系统炎症模型小鼠靶向性研究
1.给药方案
取炎症小鼠72只，随机分为2组，每组36只，给药前禁食不禁水过夜，2组荷瘤小鼠尾分别静脉注射市售左氧氟沙星以及布洛芬阳离子脂质体中性粒细胞递药系统，剂量均为布洛芬15mg/kg，分别于给药后0.167h、0.5h、1h、2h、4h、8h、12h、24h、48h、72h、96h和120h时间点，摘眼球取血后，处死小鼠，取心、肝、脾、肾、右后足跖5个组织，用生理盐水洗净，并用滤纸吸干残留生理盐水后称重。各组织按下述方法处理，用HPLC方法测各时间点的各组织样品中所含布洛芬浓度。
2.样品处理
取荷瘤小鼠各组织样品(心、肝、脾、肾、右后足跖)，称重，置于采血管中，加入2mL的生理盐水，经组织匀浆器高速分散成组织匀浆液。取200μL各组织匀浆液加入200μL乙腈，涡旋5min后，10000×g离心10min，取上清液，HPLC进样，根据线性方程计算组织样品中布洛芬的含量。
3.HPLC方法
色谱柱：
Figure PCTCN2014092340-appb-000016
ODS-SP column(250mm×4.6mm×5μm,GL Sciences Inc.,Japan)
流动相：甲醇:水＝80:20(v:v)以0.01mol/L磷酸二氢钾溶液(pH3.0)：乙腈＝45：55(v:v)
检测波长：263nm
柱温：室温
流速：1.0mL/min
进样量：20μL
组织匀浆的物质不干扰布洛芬的分离测定，布洛芬的保留时间约为10.2min。
4.小鼠体内分布结果
表13布洛芬在各脏器中分布
Figure PCTCN2014092340-appb-000017
表14布洛芬阳离子脂质体中性粒细胞递药系统在各脏器的分布
Figure PCTCN2014092340-appb-000018
由上表可知，市售布洛芬注射剂在炎症模型小鼠体内情况，肝脏分布最多，其次为心、脾脏、肾脏，炎症部位分布较少；与市售布洛芬注射剂相比，中性粒细胞递药系统分布最多的器官为炎症部位足爪和脾脏，其次为肝脏器官，在其他器官分布较少。
5.靶向性评价
应用Kinetic 4.0药代动力学程序统计方法计算静脉注射布洛芬、中性粒细胞递药系统后组织中AUC0-∞、AUC0-120h等参数，计算相对摄取率(relative ratio,Re)、靶向效率(brain targeting efficiency,Te)。
相对摄取率(relative ratio,Re)是指中性粒细胞递药系统与市售制剂布洛芬在足爪中AUC的比值，相对摄取率越大，表明该制剂相对于布洛芬在炎症组织的靶向性越强。靶向效率(brain targeting efficiency,Te)是指同一种制剂在炎症部位足爪中的AUC与其他组织中 AUC的比值，Te大于1表示该制剂对于炎症部位右后足跖的选择性大于相比较的其他组织，Te越大，则表明相对于被比较的其他组织，该制剂对非特异性炎症部位右后足跖的选择性越强。
中性粒细胞递药系统与市售制剂布洛芬相比的炎症部位Re(0-t)和Re(0-Int)如图17所示，并且其Te均大于1(图18)，说明中性粒细胞递药系统具有很好的炎症靶向性，能够快速的穿过炎症血管，将药物有效传递到炎症组织。
四、中性粒细胞递药系统对佐剂性关节炎小鼠模型药效学评价
炎症模型小鼠48只，随机分成4组，每组12只，分别为炎症模型建立后生理盐水组、单纯中性粒细胞组、市售制剂组、中性粒细胞递药系统组，其中市售制剂组的给药剂量为15mg/kg，中性粒细胞递药系统组每只静脉注射约5×106个NEs/只，给药剂量约15mg/kg。所有动物分别在术后1、2、3、4、6、8、10给药，共给药7次。同时设给予相同细胞密度，相同生理盐水体积组作为空白对照。给药后，每隔3天进行关节炎指数评分，观察每组大鼠的继发病变。
每只足爪的关节炎指数评分标准：0＝正常；1＝踝关节出现红斑和轻微肿胀；2＝踝关节到跖关节或掌关节红斑和轻微肿胀；3＝踝关节到跖趾关节或掌关节出现红斑和中度肿胀；4＝踝关节到趾关节出现红斑和重度肿胀。每只小鼠鼠最多评12分，结果如图19所示。对于关节炎指数评分出现的峰值以及出现峰值的时间，与生理盐水组相比，单纯给予中性粒细胞，两组的没有显著性差异，给予市售制剂布洛芬后，虽然出现的峰值数有所降低，出现峰值的时间有所延长，但是给予中性粒细胞递药系统后，其出现的峰值和峰值出现的时间都有的明显的下降和延长。证明本发明所制备的中性粒细胞递药系统有效的抑制变态反应性炎症的发生及发展。

Claims (12)

  1. 一种炎症靶向的中性粒细胞递药系统，其特征在于所述的递药系统由中性粒细胞以及以直接或间接地方式载到中性粒细胞内或表面的治疗性物质或者检测性物质组成。
  2. 根据权利要求1所述的炎症靶向的中性粒细胞递药系统，其特征在于所述的治疗性物质选自药物、DNA、RNA、蛋白质或多肽中的一种或多种；所述的检测性物质选自探针、显影剂中的一种或多种。
  3. 根据权利要求1或2所述的炎症靶向的中性粒细胞递药系统，其特征在于所述的间接地方式是指将纳米载体作为工具，将治疗性物质或者检测性物质先装载在纳米载体内，制成纳米制剂，再将纳米制剂荷载到中性粒细胞内或表面。
  4. 根据权利要求3所述的炎症靶向的中性粒细胞递药系统，其特征在于所述的纳米载体选自正电性、负电性或近中性的粒径为1-1000nm的纳米制剂。
  5. 根据权利要求3所述的炎症靶向的中性粒细胞递药系统，其特征在于所述的纳米载体为金纳米粒/棒、磁性纳米粒、介孔二氧化硅纳米粒、石墨烯、脂质体、胶束、纳米乳、纳米球、纳米囊、微球、微丸或树枝状聚合物。
  6. 根据权利要求2所述的炎症靶向的中性粒细胞递药系统，其特征在于所述的药物选自传出神经系统药物、中枢神经系统药、心血管系统药、激素及呼吸消化系统药、抗菌或抗病毒药物、抗肿瘤药物、维生素中的一种或多种。
  7. 根据权利要求6所述的炎症靶向的中性粒细胞递药系统，其特征在于所述的药物选自地西泮、苯妥英钠、氯丙嗪、氟西汀、美沙酮、甲氯芬酯、氯贝胆碱、硫酸阿托品、异丙肾上腺素、马来酸氯苯那敏、盐酸普鲁卡因、盐酸普萘洛尔、盐酸维拉帕米、盐酸胺碘酮、氯沙坦、硝酸甘油、多巴酚丁胺、辛伐他汀、氯吡格雷、哌唑嗪、西咪替丁、地芬尼多、西沙比利、联苯双酯、阿司匹林、吲哚美辛、盐酸氮芥、氟尿嘧啶、紫杉醇、阿莫西林、四环素、氨基糖苷、罗红霉素、氯霉素、左氟沙星、异烟肼、磺胺嘧啶、氟康唑、盐酸金刚烷胺、磷酸氟喹、格列苯脲、氢氯噻嗪、前列腺素、胰岛素、雌二醇、维生素A或维生素C中的一种或多种。
  8. 权利要求1所述的炎症靶向的中性粒细胞递药系统在制备治疗或诊断炎症和/或炎症相关疾病的药物或试剂中的应用。
  9. 根据权利要求8所述的应用，其特征在于所述的炎症包括感染性炎症、非特异性炎症、变态反应性炎症以及炎症相关性疾病。
  10. 根据权利要求9所述的应用，其特征在于所述的感染性炎症包括病毒、细菌或细菌产物造成的炎症；所述的非特异性炎症为物理性炎症，包括手术或外伤引起的红肿、疼痛；变态反应 性炎症包括狼疮性皮炎、过敏性哮喘或风湿性关节炎；炎症相关性疾病包括肿瘤术后复发的治疗、动脉粥样硬化或缺血缺氧性脑病。
  11. 一种治疗炎症和/或炎症相关疾病的药物，其特征在于包含权利要求1所述的炎症靶向的中性粒细胞递药系统。
  12. 一种诊断炎症和/或炎症相关疾病的试剂，其特征在于包含权利要求1所述的炎症靶向的中性粒细胞递药系统。
PCT/CN2014/092340 2014-09-20 2014-11-27 一种炎症靶向的中性粒细胞递药系统及其应用 WO2016041250A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US15/512,726 US11085022B2 (en) 2014-09-20 2014-11-27 Inflammation-targeted neutrophil granulocyte drug delivery system and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410484577.4A CN104225609B (zh) 2014-09-20 2014-09-20 一种炎症靶向的中性粒细胞递药系统及其应用
CN201410484577.4 2014-09-20

Publications (1)

Publication Number Publication Date
WO2016041250A1 true WO2016041250A1 (zh) 2016-03-24

Family

ID=52215082

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2014/092340 WO2016041250A1 (zh) 2014-09-20 2014-11-27 一种炎症靶向的中性粒细胞递药系统及其应用

Country Status (3)

Country Link
US (1) US11085022B2 (zh)
CN (2) CN107812197B (zh)
WO (1) WO2016041250A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113018453A (zh) * 2021-03-19 2021-06-25 河南省人民医院 一种靶向药物共递送纳米系统及其制备方法和应用
CN114632071A (zh) * 2022-03-23 2022-06-17 长春工业大学 一种脂质体纳米粒子及其制备方法和应用

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106039317A (zh) * 2016-05-23 2016-10-26 浙江工业大学 一种中性粒细胞载药制剂及应用
CN106667990A (zh) * 2016-11-17 2017-05-17 郑州郑先医药科技有限公司 一种治疗风湿病的药物
CN107625970B (zh) * 2017-08-14 2021-03-19 山东大学齐鲁医院 一种声辐射力强化炎性细胞致动脉粥样硬化仿生微泡复合体富集与靶向系统
CN109224063B (zh) * 2018-08-31 2020-11-10 厦门大学 双重负载肽类及化疗药物的纳米复合载体及其制备与应用
CN109758424B (zh) * 2019-02-26 2021-01-29 李翠 一种消化系统炎症药物靶向运输药物系统
CN111000822B (zh) * 2019-11-11 2021-09-03 沈阳药科大学 阿霉素-吲哚菁绿仿生纳米颗粒及其用途
CN111701830A (zh) * 2020-06-24 2020-09-25 宁波计氏金属新材料有限公司 一种金属喷涂处理工艺
CN112472685B (zh) * 2020-12-10 2023-03-24 哈尔滨工业大学 一种杂化中性粒细胞机器人的制备方法
CN114642736B (zh) * 2020-12-17 2023-12-12 中国科学院深圳先进技术研究院 一种透血脑屏障药物递送系统及其制备方法和应用
CN113952461B (zh) * 2021-09-07 2024-03-22 浙江大学 一种仿中性粒细胞纳米递药系统及其制备方法和应用
CN114558125B (zh) * 2021-12-30 2024-02-20 厦门大学附属心血管病医院 一种尿素酶驱动中性粒细胞递药系统及其合成方法
WO2023133634A1 (en) * 2022-01-11 2023-07-20 Centre For Commercialization Of Regenerative Medicine Loaded cell populations, methods of preparation, and methods of use thereof
WO2023245074A2 (en) * 2022-06-14 2023-12-21 Purdue Research Foundation Car-expressing pluripotent stem cell-derived neutrophils loaded with drug nanoparticles and uses thereof
CN116617408B (zh) * 2023-03-31 2023-12-08 江南大学附属医院 一种微生物与功能核酸共递送系统及其制备方法与应用
CN116850248B (zh) * 2023-08-22 2024-03-19 通化万通药业股份有限公司 咳克平胶囊及其制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040060A1 (en) * 1995-06-07 1996-12-19 President And Fellows Of Harvard College Intracellular delivery of macromolecules
WO2011046842A1 (en) * 2009-10-12 2011-04-21 The Regents Of The University Of California Targeted nanoclusters and methods of their use

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CH693905A5 (de) 1999-04-15 2004-04-15 Eprova Ag Stabile kristalline Salze von 5-Methyltetrahydrofolsäure.
US6680068B2 (en) * 2000-07-06 2004-01-20 The General Hospital Corporation Drug delivery formulations and targeting
US20040210289A1 (en) * 2002-03-04 2004-10-21 Xingwu Wang Novel nanomagnetic particles
MXPA06011171A (es) * 2004-06-15 2007-01-25 Baxter Int Aplicacion ex-vivo de agentes terapeuticos microparticulados solidos.
US10952965B2 (en) * 2009-05-15 2021-03-23 Baxter International Inc. Compositions and methods for drug delivery

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040060A1 (en) * 1995-06-07 1996-12-19 President And Fellows Of Harvard College Intracellular delivery of macromolecules
WO2011046842A1 (en) * 2009-10-12 2011-04-21 The Regents Of The University Of California Targeted nanoclusters and methods of their use

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JING QIN ET AL.: "Surface Modification of RGD-Liposomes for Selective Drug Delivery to Monocytes/Neutrophils in Brain", CHEM. PHARM. BULL., vol. 55, no. 8, 31 December 2007 (2007-12-31), pages 1192 - 1197, XP055018501, DOI: doi:10.1248/cpb.55.1192 *
QIN, JING ET AL.: "Studies on RGD-Mediated Brain-Target Ferulic Acid Liposome", CDFD MEDICAL SCIENCE AND TECHNOLOGY COMPILES, vol. 9, 15 September 2013 (2013-09-15) *
SANYOG JAIN ET AL.: "RGD-anchored magnetic liposome for monocytes/neutrophils-mediated brain targeting", INTERNATIONAL JOURNAL OF PHARMACEUTICS, vol. 26, 31 December 2003 (2003-12-31), pages 43 - 55 *
WANG, SHUANG ET AL.: "Progress in Drug Delivery System for Brain Targeting", CHINESE JOURNAL OF PHARMACEUTICALS, vol. 43, no. 12, 31 December 2012 (2012-12-31), pages 137 - 142 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113018453A (zh) * 2021-03-19 2021-06-25 河南省人民医院 一种靶向药物共递送纳米系统及其制备方法和应用
CN114632071A (zh) * 2022-03-23 2022-06-17 长春工业大学 一种脂质体纳米粒子及其制备方法和应用

Also Published As

Publication number Publication date
CN107812197B (zh) 2018-09-18
CN104225609B (zh) 2018-05-18
US20170292113A1 (en) 2017-10-12
CN104225609A (zh) 2014-12-24
CN107812197A (zh) 2018-03-20
US11085022B2 (en) 2021-08-10

Similar Documents

Publication Publication Date Title
WO2016041250A1 (zh) 一种炎症靶向的中性粒细胞递药系统及其应用
Carvalho et al. Advances and challenges in nanocarriers and nanomedicines for veterinary application
Singh et al. Advances in nanotechnology-based carrier systems for targeted delivery of bioactive drug molecules with special emphasis on immunotherapy in drug resistant tuberculosis–a critical review
JP6392209B2 (ja) 粘液内層を通過する迅速な透過のための脂質ベース薬物キャリア
Zarif et al. Cochleates: new lipid-based drug delivery system
KR20140138639A (ko) 점막 침투 강화를 나타내는 나노 입자 제형
WO2010083778A1 (zh) 注射用肺靶向脂质体药物组合物
CN107157950B (zh) 一种白蛋白纳米粒及其制备方法和用途
CN111920768A (zh) 一种包载分子靶向药物脂质体及其在制备治疗肿瘤药物中的应用
Liu et al. Enhanced treatment for cerebral ischemia-reperfusion injury of puerarin loading liposomes through neutrophils-mediated targeted delivery
CN113827738B (zh) 唾液酸修饰地塞米松棕榈酸酯脂质体及其制备和应用
Ma et al. Hydroxycamptothecin (HCPT)-loaded PEGlated lipid–polymer hybrid nanoparticles for effective delivery of HCPT: QbD-based development and evaluation
CN102379850B (zh) 一种穿越人体粘液屏障的靶向给药脂质体
Hwang Targeted delivery of erythropoietin hybridized with magnetic nanocarriers for the treatment of central nervous system injury: A literature review
WO2022068242A1 (zh) 一种雷帕霉素组合物及其制备方法
Wang et al. Temozolomide hexadecyl ester targeted plga nanoparticles for drug-resistant glioblastoma therapy via intranasal administration
CN102370623A (zh) 经嗅区通路递送脑内药物的液态脂质微粒、制备方法及其制剂
Zou et al. Unleashing the potential: integrating nano-delivery systems with traditional Chinese medicine
CN113768878A (zh) 一种榄香烯卡巴他赛双靶向仿生脂质体及其制备方法和应用
CN105997872B (zh) 一种含有泊沙康唑的眼用纳米胶束抗真菌溶液
Liu et al. Targeted Delivery of Macrophage Membrane Biomimetic Liposomes Through Intranasal Administration for Treatment of Ischemic Stroke
CN105287612B (zh) 共载盐霉素钠与阿霉素纳米脂质体及其制备方法与应用
CN101152545A (zh) 黄姜水溶性皂苷脂质体及其制备方法和用途
CA3176169A1 (en) Compositions and methods for treating cytokine storms
Wang et al. A highly atom-economical bioactive nanocarrier for synergistically enhanced antitumor with reduced liver injury

Legal Events

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

Ref document number: 14901822

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 15512726

Country of ref document: US

122 Ep: pct application non-entry in european phase

Ref document number: 14901822

Country of ref document: EP

Kind code of ref document: A1