WO2022088708A1 - Schisanlactone e targeted drug loading system, and preparation method therefor and application thereof - Google Patents

Schisanlactone e targeted drug loading system, and preparation method therefor and application thereof Download PDF

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WO2022088708A1
WO2022088708A1 PCT/CN2021/101479 CN2021101479W WO2022088708A1 WO 2022088708 A1 WO2022088708 A1 WO 2022088708A1 CN 2021101479 W CN2021101479 W CN 2021101479W WO 2022088708 A1 WO2022088708 A1 WO 2022088708A1
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prussian blue
nanoparticles
membrane
loaded
cytonin
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PCT/CN2021/101479
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French (fr)
Chinese (zh)
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王炜
余黄合
刘斌
蔡雄
李斌
彭彩云
江星明
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湖南中医药大学
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    • 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
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/57—Magnoliaceae (Magnolia family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/79—Schisandraceae (Schisandra family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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]

Definitions

  • the invention relates to a targeted drug, in particular to a cytoplasmin targeted drug-carrying system.
  • the invention also relates to a preparation method of a cytosine targeted drug-carrying system and an application of the cytosine targeted drug-carrying system in the preparation of an anti-rheumatoid arthritis drug.
  • Blood tube is a commonly used medicinal plant of the Tujia family. Blood tube is sweet, slightly acrid, warm. It has the functions of invigorating blood and promoting blood circulation, expelling wind and removing dampness, and promoting qi and relieving pain. It is often used by Tujia folks to treat rheumatic arthralgia, epigastric pain, menstrual pain, bone pain, rheumatoid arthritis, lumbar muscle strain, cold, and postpartum rheumatic paralysis. Cytokinin is a triterpenoid pentalactone E (schisanlactone E, SE) extracted from blood vessels.
  • schisanlactone E, SE triterpenoid pentalactone E
  • SE can inhibit the reproduction and growth of rheumatoid arthritis fibroblast-like synovial cells, and inhibit the The expression of inflammatory factors can effectively inhibit the incidence of adjuvant-induced arthritis in rats, inhibit the swelling of paws, protect the joint tissue of rats, and have the effect of resisting rheumatoid arthritis (RA).
  • Rheumatoid arthritis is a chronic, inflammatory synovitis-based systemic disease of unknown etiology. It is characterized by polyarticular, symmetrical and aggressive joint inflammation of the facet joints of the hands and feet, often accompanied by extra-articular organ involvement and positive serum rheumatoid factor, which can lead to joint deformity and loss of function, resulting in loss of labor or disability. Rheumatoid arthritis is currently incurable and requires long-term treatment. However, long-term drug treatment usually produces strong adverse reactions, which reduces the patient's tolerance to drugs and affects the effect of long-term treatment of rheumatoid arthritis.
  • the technical problem to be solved by the present invention is to provide a targeted drug-carrying system for cytosine, which can deliver cytosine to the diseased part of rheumatoid arthritis and increase the drug concentration in the diseased part.
  • the further technical problem to be solved by the present invention is to provide a preparation method of a cytosine targeted drug-carrying system, so as to obtain a targeted drug capable of delivering cytosine to the lesions of rheumatoid arthritis.
  • the technical problem to be solved by the present invention is to provide the application of the cytonin-targeted drug-carrying system in the preparation of anti-rheumatoid arthritis drugs.
  • one aspect of the present invention provides a cytosine targeted drug-loading system, comprising cytosine, Prussian blue nanoparticles, a biomimetic membrane coating layer and a hyaluronic acid modified layer.
  • Described hemoglobin has the structure shown in following formula (I):
  • the Prussian blue supporting layer is Prussian blue cubic nanoparticles, Prussian blue spherical particles or a mixture of Prussian blue cubic nanoparticles and Prussian blue spherical particles.
  • Prussian blue (PB) a nanomaterial with a metal-organic framework (MOF) structure
  • FDA U.S. Food and Drug Administration
  • the metal-organic framework structure of the cube-shaped Prussian blue nanoparticles can better combine with the cytosine to carry the cytosine to the lesions of rheumatoid arthritis.
  • the biomimetic membrane wrapping layer is a fusion membrane composed of red blood cell membrane and rheumatoid arthritis fibroblast-like synovial cell membrane.
  • Red blood cell membrane RBCM
  • Rheumatoid arthritis fibroblast-like synovial membrane RAFLSM
  • CD44 molecules protein molecules with special functions, such as CD44 molecules, which can target the inflammatory synovial tissue in the pathological state of arthritis. This is a special "homing effect”.
  • the biomimetic membrane which is a hybrid of erythrocyte membrane and rheumatoid arthritis fibroblast-like synovial membrane, has the functional characteristics of two biofilms. After encapsulating the drug material, the drug material can be accurately delivered to the site of arthritis. .
  • the erythrocyte membrane is a rat erythrocyte membrane.
  • Rat erythrocyte membrane is easy to obtain and has good biocompatibility.
  • a second aspect of the present invention provides a method for preparing a cytosine targeted drug-carrying system, comprising the following steps: S10, synthesizing Prussian blue nanoparticles; S20, preparing a biomimetic membrane; S30, using the Prussian blue nanoparticles and Cytolin is used to prepare Prussian blue-loaded cytolin nanoparticles; S40, use the biomimetic membrane and the Prussian blue-loaded cytolin nanoparticles to prepare a biomimetic membrane to encapsulate the Prussian blue-loaded cytolin nanoparticles; S50, to all The biomimetic membrane wraps the Prussian blue-loaded cytosine nanoparticles for hyaluronic acid modification to obtain the cytosine targeted drug-loading system.
  • the preparation method of the biomimetic membrane includes the following steps: S21, after hemolyzed rat erythrocytes, ultrasonically disrupted and filtered to obtain erythrocyte membrane fragments; S22, hypotonic rheumatoid arthritis fibroblast-like synovial cells After lysis, ultrasonically disrupted, freeze-thawed several times, and centrifuged to obtain rheumatoid arthritis fibroblast-like synovial cell fragments; S23, equalize the volumes of the red blood cell membrane fragments and the rheumatoid arthritis fibroblast-like synovial cell fragments Mixing, pressure filtration, and centrifugation obtain the biomimetic membrane.
  • the preparation method of the Prussian blue-loaded cytolin nanoparticles is as follows: mixing the cytidine and the Prussian blue nanoparticles in a mass ratio of 1:1-4, stirring, and dialysis to remove the unloaded cytidine to obtain the Prussian blue-loaded cytoplasmin nanoparticles.
  • the preparation method of the biomimetic membrane encapsulating the Prussian blue-loaded cytonin nanoparticles is as follows: mixing the biomimetic membrane and the Prussian blue-loaded cytonin nanoparticles in equal volumes, and stirring, so that the biological
  • the biomimetic membrane fully wraps the Prussian blue-loaded cytonin nanoparticles, centrifuged to collect the precipitate, and resuspended, to obtain the biomimetic membrane-wrapped Prussian blue-loaded cytonin nanoparticles.
  • the method for modifying hyaluronic acid comprises the following steps: S51, adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinic acid to hyaluronic acid The imide is stirred and activated; S52, activated hyaluronic acid and phospholipid-polyethylene glycol-amino group are added to the biomimetic membrane-wrapped Prussian blue-loaded cytolin nanoparticles, and stirred; S53, dialysis using a dialysis bag, The hemocystin targeted drug-loading system is obtained.
  • the third aspect of the present invention provides the application of the cytosine targeted drug-carrying system in the preparation of an anti-rheumatoid arthritis drug.
  • the cytosine targeted drug-loading system of the present invention can target and accumulate into the diseased RAFLS cells, increase the cytosine concentration in the diseased part, and enhance the therapeutic effect. At the same time, it can also reduce the concentration of cytosine in the blood and other parts of the body, and reduce the adverse reactions caused by cytosine.
  • the preparation method of the cytosine targeted drug-carrying system of the present invention provides a preparation method of the cytosine targeted drug-carrying system, which can realize the targeted treatment of rheumatoid arthritis.
  • a variety of suitable pharmaceutical dosage forms can be prepared, which can be used in the treatment of rheumatoid arthritis.
  • Fig. 1 is the scanning electron microscope photograph of PB NPs of an embodiment of the present invention
  • Fig. 2 is the PB NPs transmission electron microscope photograph of an embodiment of the present invention
  • FIG. 3 is a schematic diagram of the formation of a RAFLSM structure according to an embodiment of the present invention.
  • FIG. 4 is a schematic diagram of the ultraviolet absorption characterization of RBCM, RAFLSM and RFM according to an embodiment of the present invention
  • Fig. 5 is the transmission electron microscope photograph of HA@RFM@PB@SE NPs obtained by an embodiment of the present invention
  • FIG. 6 is a schematic diagram of a particle size distribution curve of each nanoparticle in an embodiment of the present invention.
  • FIG. 7 is a schematic diagram of the zeta potential of each nanoparticle in an embodiment of the present invention.
  • FIG. 8 is a schematic diagram of rat hemolysis of each nanoparticle in an embodiment of the present invention.
  • FIG. 9 is a schematic diagram of the effect of each nanoparticle on the cell viability of RAFLS cells in an embodiment of the present invention.
  • Figure 10 is a fluorescence imaging diagram of the distribution of each nanoparticle in a rat in an embodiment of the present invention.
  • Figure 11 is an in vitro fluorescence imaging image of the distribution of each nanoparticle in rat viscera in one embodiment of the present invention.
  • RAFLS cells were purchased from Beijing Beina Chuanglian Institute of Biotechnology; SPF SD rats were purchased from Hunan Slike Jingda Laboratory Animal Co., Ltd. (certificate number: 43004700063752).
  • potassium ferricyanide K 3 [Fe(CN) 6 ].3H 2 O
  • PVP poly(N-vinylpyrrolidone)
  • 1 ⁇ PBS phosphate buffered saline solution
  • 0.25 ⁇ PBS were purchased from Gibco, USA
  • Cell Membrane Protein Extraction Kit Solution A and phenylmethylsulfonyl fluoride (PMSF) were purchased from Beyotime Institute of Biotechnology , fetal bovine serum (FBS), DMEM medium and trypsin cell digestion solution were purchased from Gibco, USA
  • 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N- Hydroxysuccinimide (NHS) was purchased from Sigma-Aldrich Life Science Technology Co., Ltd.
  • phospholipid-polyethylene glycol-amino (DSPE-PEG2000-NH 2 ) was purchased from Shanghai Pengsheng Biotechnology Co., Ltd.; DMSO, MTT cells Proliferation and cytotoxicity detection kits were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; heat-killed Mycobacterium tuberculosis H37Ra (Sigma Aldrich, USA, 20150411).
  • Other drugs and reagents are conventional commercially available products.
  • the instruments and instruments used, the scanning electron microscope was produced by JEOL's ⁇ , model: JSM-6700F; the ultrasonic cell disruptor was produced by Nanjing Saifei Biotechnology Co., Ltd., model: Biosafer 900-92; ultra-pure
  • the water preparation system is produced by Veolia ELGA Water Treatment Technology (Shanghai) Co., Ltd., model: FLB00003057; the Zeta potential and nanoparticle size analyzer is produced by Malvin Instrument Co., Ltd., UK, model: Zetasizer Nano; the filter is produced by Merck Libo Life Technology Co., Ltd.; dialysis bag by Beijing Soleibao Technology Co., Ltd.; high-speed refrigerated centrifuge (Eppendorf 5810R, Germany). Others are common commercially available products.
  • the ultrapure sterile water used in the embodiment of the present invention is 18.2M ⁇ ultrapure water prepared by an ultrapure water preparation system, and the pH is 7.4.
  • the crude PB nanomaterials were dispensed into 1.5mL EP tubes and centrifuged at 13500rpm for 15 minutes at 4°C. After centrifugation, discard the supernatant, combine the materials in each two EP tubes into one tube, add 1 mL of sterile deionized water, mix thoroughly, centrifuge under the same conditions for 15 minutes, and then add 1 mL of sterile deionized water to each tube.
  • the EP tube was concentrated into 1 tube to obtain refined PB material.
  • the obtained PB nanoparticles are cubic nanoparticles with a particle size of 70-80 nm.
  • the obtained PB nanoparticles were photographed by a transmission electron microscope, and the transmission electron microscope photograph as shown in FIG. 2 was obtained.
  • the obtained refined PB materials are cubic PB nanoparticles.
  • FBS fetal bovine serum
  • Ultrasonic disruption was performed using an ultrasonic cell disruptor for 5 minutes, with a disruption power of 80W, each sonication was disrupted for 0.3 seconds, and the cells were stopped for 0.5 seconds. After crushing, it was quickly frozen at -80°C, and the freeze-thaw was repeated 3 times. The melted liquid was centrifuged at 800 rpm for 10 minutes at 4°C, the supernatant was further centrifuged at 12,000 rpm at 4°C for 30 minutes, and the precipitate was collected to obtain cell membrane fragments.
  • RBCM and RAFLSM in equal volume, for example: take 600 ⁇ L of RBCM and 600 ⁇ L of RAFLSM, mix together, sonicate for 5 minutes at a constant temperature of 37 °C, and stir at 800 rpm for 4 hours.
  • the mixtures were extruded through 1 ⁇ m, 400 nm and 200 nm polycarbonate porous membranes, respectively, to facilitate membrane fusion.
  • the mixture was then centrifuged at 21,000 rpm for 30 minutes at 4°C to collect the precipitate, which is RBC&RAFLS biomimetic membrane vesicles, and resuspended in 1 ⁇ PBS.
  • the obtained RFM is a hybrid membrane formed by alternately connecting RBCM and RAFLSM. Take 10 ⁇ L of the extracted RBCM, RAFLSM and the obtained RFM respectively, add 290 ⁇ L of 1 ⁇ PBS, mix well, add to the cuvette with a pipette, observe the ultraviolet characteristic absorption peaks of the three membranes with an ultraviolet spectrophotometer, and obtain The results are shown in Figure 4.
  • the UV absorption curve of RBCM has characteristic absorption peaks at 210nm and 250-275nm
  • the UV absorption curve of RAFLSM has characteristic absorption peaks at 405nm and 540-560nm
  • the UV absorption curve of RFM has the same characteristics as RBCM.
  • the characteristic absorption peaks superimposed with the ultraviolet absorption curve of RAFLSM that is, there are characteristic absorption peaks at the wavelengths of 210nm, 250-275nm, 405nm and 540-560nm. From this, it can be seen that the RFM has both the RBCM segment and the RAFLSM segment.
  • RFM RBC&RAFLS biomimetic membrane
  • HA@RFM@PB@SE NPs (0.01mg/mL) solution and drop it on a copper mesh covered with carbon film, put it under an infrared lamp to dry at a temperature of 60 °C, and use a transmission electron microscope to photograph its shape and RFM film Status of the package.
  • the HA@RFM@PB@SE NPs displayed a typical core-shell structure.
  • the particle size of RFM@PB@SE NPs is about 140 nm, which shows that the particle size of nanoparticles increases significantly after film encapsulation, and the increased particle size is also consistent with the thickness of one layer of RFM.
  • the particle size of HA@RFM@PB@SE NPs is about 160 nm, indicating that the HA-targeted material further increases the particle size of nanoparticles after HA modification. As shown in Fig.
  • the zeta potentials of PB NPs, PB@SE NPs, RFM@PB@SE NPs and HA@RFM@PB@SE NPs are -16.1mV, -5.4mV, -23.6mV and -15.3mV, respectively.
  • the Zeta potential of RFM@PB@SE NPs and HA@RFM@PB@SE NPs is close to that of HA@RFM@PB@SE NPs after wrapping RFM, and the absolute increase of their surface charges may be related to the charge shielding effect produced by RFM vesicle wrapping.
  • the addition of positively charged PEG to HA reduces the Zeta potential of the nanoparticle surface relatively, which also confirms the successful preparation of HA@RFM@PB@SE NPs from another aspect.
  • Hemolysis(%) (I/I 0 ) ⁇ 100%, where Hemolysis is the hemolysis rate, I represents the absorbance of the test sample, and I 0 represents the absorbance of the positive control (100% hemolysis). Each sample was repeated three times, and the average value was taken as the hemolysis rate of the sample.
  • the detection results are shown in Figure 8. As shown in FIG.
  • RAFLSs cells use 100 U/mL penicillin and 100 ⁇ g/mL streptomycin as double antibodies, inoculate into high glucose DMEM medium containing 10% FBS, and place them in an incubator containing 5% CO at 37°C Culture, and after the cells adhere to the wall and grow well, passage once every 2 to 3 days.
  • RAFLS cells Take the RAFLS cells in logarithmic growth phase, wash twice with 1 ⁇ PBS, add 500 ⁇ L of 0.25% trypsin cell digestion solution and place them in a 37°C incubator to digest for 4 minutes, and then use 1 mL of DMEM medium containing 10% FBS to stop the digestion , the cells were collected, placed in a 1.5 mL EP tube, centrifuged at 900 rpm for 5 minutes, the supernatant was removed, and 1 mL of DMEM medium containing 10% FBS was added to prepare a single cell suspension.
  • the positive drug group was added with a final concentration of 5 ⁇ M methotrexate and indomethacin, and PB NPs with a final PB concentration of 25 ⁇ g/mL, SE with a final SE concentration of 4.5 ⁇ M, PB@SE NPs, RFM@PB@SE NPs and HA were added to the other wells, respectively.
  • @RFM@PB@SE NPs Among them, PB NPs, PB@SE NPs, RFM@PB@SE NPs and HA@RFM@PB@SE NPs were each set up in two groups, with a total of 12 groups, each with 6 duplicate wells.
  • PB@SE NPs After irradiating each well of a group of PB NPs, PB@SE NPs, RFM@PB@SE NPs, and HA@RFM@PB@SE NPs with 808 nm near-infrared laser (1 W/cm 2 ) for 5 min, respectively, The 96-well plate was placed in a 5% CO2 incubator at 37°C for 48 hours, the medium was discarded, and the cell growth state was observed. Incubate with serum-free DMEM containing 10% MTT for 4 hours, discard the supernatant, add 100 ⁇ L of DMSO to each well, and detect the OD value at 492 nm on a microplate reader.
  • the inhibition rates of PB NPs, SE, PB@SE NPs, RFM@PB@SE NPs, and HA@RFM@PB@SE NPs groups to RAFLS cells were 12.95%, 27.25%, and 25.02, respectively. %, 51.58%, 70.57%.
  • the inhibition rates of the PB NPs+Laser, PB@SE NPs+Laser, RFM@PB@SE NPs+Laser and HA@RFM@PB@SE NPs+Laser groups on RAFLS cells were 28.60%, 32.02%, 28.60%, 32.02%, respectively.
  • RFM@PB@SE NPs formed by encapsulating PB@SE NPs by RFM and HA@RFM@PB@SE NPs, the drug-carrying system for cylindrokinin of the present invention, have significantly higher inhibition rates on RAFLS cells than pure SE and PB@ SE NPs.
  • the inhibition rate of HA@RFM@PB@SE NPs of the present invention on RAFLS cells reaches and exceeds the level of positive drugs methotrexate and indomethacin (5 ⁇ M methotrexate and indomethacin on RAFLS cells).
  • the inhibition rates were 70.81% and 59.48%, respectively), especially, after 808 nm near-infrared laser irradiation, the inhibition rate of the HA@RFM@PB@SE NPs of the present invention on RAFLS cells was significantly greater than that of methotrexate and indomethacin Xin, has better anti-rheumatoid arthritis effect.
  • SPF grade SD rats with a body weight of 70-90 g were taken and divided into 4 groups with 6 rats in each group, and were raised in the IVC barrier system to maintain constant temperature and humidity.
  • 0.1 mL of complete Freund's adjuvant (CFA) containing 200 ⁇ g of heat-killed Mycobacterium tuberculosis (Mtb) was subcutaneously injected into the base of the rat's tail to establish an AIA model.
  • CFA complete Freund's adjuvant
  • Mtb heat-killed Mycobacterium tuberculosis
  • N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and ICG were added to 1 ⁇ PBS, respectively, to form NHS with a content of 1 mg/mL, EDC content 1 mg/mL, ICG content 2.5 mg/mL solution.
  • NHS N-hydroxysuccinimide
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • ICG ICG
  • NHS, EDC, and ICG were added to PB@SE NPs, respectively, to form solutions with NHS content of 1 mg/mL, EDC content of 1 mg/mL, ICG content of 2.5 mg/mL, and SE content of 0.21 mg/mL.
  • a dialysis bag with a molecular weight cut-off of 3.5 KDa was used for 24 h to remove free ICG to obtain ICG-PB@SE NPs.
  • ICG-RFM@PB@SE NPs and ICG-HA@RFM@PB@SE NPs with SE content of 0.21 mg/mL were obtained, respectively.
  • mice The prepared ICG-PBS, ICG-PB@SE NPs, ICG-RFM@PB@SE NPs, and ICG-HA@RFM@PB@SE NPs were injected into one mouse via the tail vein at a volume of 1 mL/Kg, respectively. in mice.
  • swollen toes and major organs (heart, liver, spleen, lungs, kidneys) of rats were carefully collected 48 hours after injection and imaged using the Kodak Multimodal Imaging System.
  • the cytonin-targeted drug-loading system of the present invention can make the anti-rheumatoid arthritis drug SE accumulate in the lesions of rheumatoid arthritis, increase the concentration of SE in the lesions, and reduce the concentration of SE in other tissues and organs of the body. SE concentration, thereby improving the therapeutic effect of SE on rheumatoid arthritis and reducing the side effects of SE on other parts of the body.
  • the targeted drug-carrying system of cytolin of the present invention can target and combine with the rheumatoid arthritis fibroblast-like synovial cells in the rheumatoid arthritis lesions, so as to improve the blood vessels in the rheumatoid arthritis lesions.
  • the concentration of the hormone enhances the inhibitory effect on RAFLS cells, thereby improving the therapeutic effect on rheumatoid arthritis.
  • the cytosine targeted drug-carrying system of the present invention has good biocompatibility, can effectively accumulate in the rheumatoid arthritis lesions, reduces the cytosine concentration in other parts of the body, has high safety in use, and has low side effects, and can be used for Preparation of anti-rheumatoid arthritis drugs.

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Abstract

Provided is a schisanlactone E targeted drug loading system, comprising schisanlactone E, Prussian blue nanoparticles, a biomimetic membrane wrapping layer, and a hyaluronic acid modification layer. Also disclosed are a preparation method for the targeted drug loading system, and an application of the schisanlactone E targeted drug loading system in preparation of drugs for resisting rheumatoid arthritis.

Description

血筒素靶向载药体系、制备方法及其应用Cytokinin targeted drug-carrying system, preparation method and application thereof
相关申请的交叉引用CROSS-REFERENCE TO RELATED APPLICATIONS
本申请要求2020年10月28日提交的中国专利申请202011170878.1的权益，该申请的内容通过引用被合并于本文。This application claims the benefit of Chinese patent application 202011170878.1 filed on October 28, 2020, the contents of which are incorporated herein by reference.
技术领域technical field
本发明涉及靶向药物，具体涉及一种血筒素靶向载药体系。本发明还涉及一种血筒素靶向载药体系的制备方法及该血筒素靶向载药体系在制备抗类风湿关节炎药物中的应用。The invention relates to a targeted drug, in particular to a cytoplasmin targeted drug-carrying system. The invention also relates to a preparation method of a cytosine targeted drug-carrying system and an application of the cytosine targeted drug-carrying system in the preparation of an anti-rheumatoid arthritis drug.
背景技术Background technique
血筒是一种土家族常用的药用植物,为五味子科南五味子属植物异型南五味子Kadsuraheteroclita(Roxb)Craib，常用其藤茎入药。血筒性味甘，微辛，温。具有补血活血、祛风除湿、行气止痛之功效，土家民族民间常用于治疗风湿痹痛、胃脘痛、经痛、骨痛、风湿性关节炎、腰肌劳损、感冒、产后风瘫等症。血筒素是从血筒中提取的三萜类化合物五内酯E(schisanlactone E、SE)，现有的研究认为，SE能够抑制类风湿关节炎成纤维样滑膜细胞的繁殖和生长，并抑制其炎性因子的表达，并能有效抑制佐剂诱导的大鼠关节炎的发生率，抑制足爪肿胀，保护大鼠关节组织，具有抗类风湿关节炎(rheumatoid arthritis,RA)的作用。Blood tube is a commonly used medicinal plant of the Tujia family. Blood tube is sweet, slightly acrid, warm. It has the functions of invigorating blood and promoting blood circulation, expelling wind and removing dampness, and promoting qi and relieving pain. It is often used by Tujia folks to treat rheumatic arthralgia, epigastric pain, menstrual pain, bone pain, rheumatoid arthritis, lumbar muscle strain, cold, and postpartum rheumatic paralysis. Cytokinin is a triterpenoid pentalactone E (schisanlactone E, SE) extracted from blood vessels. Existing studies believe that SE can inhibit the reproduction and growth of rheumatoid arthritis fibroblast-like synovial cells, and inhibit the The expression of inflammatory factors can effectively inhibit the incidence of adjuvant-induced arthritis in rats, inhibit the swelling of paws, protect the joint tissue of rats, and have the effect of resisting rheumatoid arthritis (RA).
类风湿关节炎是一种病因未明的慢性、以炎性滑膜炎为主的系统性疾病。其特征是手、足小关节的多关节、对称性、侵袭性关节炎症，经常伴有关节 外器官受累及血清类风湿因子阳性，可以导致关节畸形及功能丧失，导致患者丧失劳动力或者致残。类风湿关节炎目前尚无法治愈，需要进行长期的治疗。但长期的药物治疗通常会产生较强的不良反应，降低了病人对药物的耐受和影响了类风湿关节炎的长期治疗的效果。Rheumatoid arthritis is a chronic, inflammatory synovitis-based systemic disease of unknown etiology. It is characterized by polyarticular, symmetrical and aggressive joint inflammation of the facet joints of the hands and feet, often accompanied by extra-articular organ involvement and positive serum rheumatoid factor, which can lead to joint deformity and loss of function, resulting in loss of labor or disability. Rheumatoid arthritis is currently incurable and requires long-term treatment. However, long-term drug treatment usually produces strong adverse reactions, which reduces the patient's tolerance to drugs and affects the effect of long-term treatment of rheumatoid arthritis.
发明内容SUMMARY OF THE INVENTION
本发明所要解决的技术问题是提供一种血筒素靶向载药体系，能够将血筒素递送到类风湿关节炎的病变部位，提高病变部位的药物浓度。The technical problem to be solved by the present invention is to provide a targeted drug-carrying system for cytosine, which can deliver cytosine to the diseased part of rheumatoid arthritis and increase the drug concentration in the diseased part.
本发明进一步要解决的技术问题是提供一种血筒素靶向载药体系的制备方法，得到能够将血筒素递送到类风湿关节炎的病变部位的靶向药物。The further technical problem to be solved by the present invention is to provide a preparation method of a cytosine targeted drug-carrying system, so as to obtain a targeted drug capable of delivering cytosine to the lesions of rheumatoid arthritis.
本发明还要解决的技术问题是提供血筒素靶向载药体系在制备抗类风湿关节炎药物中的应用。The technical problem to be solved by the present invention is to provide the application of the cytonin-targeted drug-carrying system in the preparation of anti-rheumatoid arthritis drugs.
为了实现上述目的，本发明一方面提供了一种血筒素靶向载药体系，包括血筒素、普鲁士蓝纳米颗粒、生物仿生膜包裹层和透明质酸修饰层。In order to achieve the above object, one aspect of the present invention provides a cytosine targeted drug-loading system, comprising cytosine, Prussian blue nanoparticles, a biomimetic membrane coating layer and a hyaluronic acid modified layer.
所述血筒素具有如下式(I)所示的结构:Described hemoglobin has the structure shown in following formula (I):
Figure PCTCN2021101479-appb-000001
Figure PCTCN2021101479-appb-000001
根据本发明，所述普鲁士蓝负载层为普鲁士蓝立方体纳米颗粒、普鲁士蓝球形颗粒或者普鲁士蓝立方体纳米颗粒与普鲁士蓝球形颗粒的混合物。普鲁士蓝(PB)是一种具有金属有机骨架(MOF)结构的纳米材料，已被美国 食品和药品监督局(FDA)批准用于治疗铊和铯放射性暴露。立方体的普鲁士蓝纳米颗粒的金属有机骨架结构能够与血筒素较好的结合，将血筒素携带到类风湿关节炎的病变部位。According to the present invention, the Prussian blue supporting layer is Prussian blue cubic nanoparticles, Prussian blue spherical particles or a mixture of Prussian blue cubic nanoparticles and Prussian blue spherical particles. Prussian blue (PB), a nanomaterial with a metal-organic framework (MOF) structure, has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of thallium and cesium radioactive exposure. The metal-organic framework structure of the cube-shaped Prussian blue nanoparticles can better combine with the cytosine to carry the cytosine to the lesions of rheumatoid arthritis.
根据本发明，所述生物仿生膜包裹层为红细胞膜与类风湿关节炎成纤维样滑膜细胞膜组成的融合膜。红细胞膜(RBCM)，具有生物相容性好等特点，可以帮助包裹的材料在血液中的运输。类风湿关节炎成纤维样滑膜细胞膜(RAFLSM)提取自关节炎症状态下的滑膜细胞，该细胞膜具有特殊功能的蛋白分子如CD44分子，能与关节炎病理状态的炎性滑膜组织进行靶向识别，进而聚集到病变的关节炎滑膜中，这是一种特殊的“归巢效应”。而红细胞膜与类风湿关节炎成纤维样滑膜细胞膜杂化成的生物仿生膜(RFM)具有两种生物膜的功能特点，在包裹了药物材料后，能够将药物材料准确递送至关节炎发病部位。According to the present invention, the biomimetic membrane wrapping layer is a fusion membrane composed of red blood cell membrane and rheumatoid arthritis fibroblast-like synovial cell membrane. Red blood cell membrane (RBCM), which has the characteristics of good biocompatibility, can help the transport of the wrapped material in the blood. Rheumatoid arthritis fibroblast-like synovial membrane (RAFLSM) is extracted from synovial cells in the state of joint inflammation. The cell membrane has protein molecules with special functions, such as CD44 molecules, which can target the inflammatory synovial tissue in the pathological state of arthritis. This is a special "homing effect". The biomimetic membrane (RFM), which is a hybrid of erythrocyte membrane and rheumatoid arthritis fibroblast-like synovial membrane, has the functional characteristics of two biofilms. After encapsulating the drug material, the drug material can be accurately delivered to the site of arthritis. .
根据本发明，所述红细胞膜为大鼠红细胞膜。大鼠红细胞膜获取容易，生物相容性较好。According to the present invention, the erythrocyte membrane is a rat erythrocyte membrane. Rat erythrocyte membrane is easy to obtain and has good biocompatibility.
本发明第二方面提供了一种血筒素靶向载药体系的制备方法，包括如下步骤：S10、合成普鲁士蓝纳米颗粒；S20、制备生物仿生膜；S30、使用所述普鲁士蓝纳米颗粒和血筒素制备普鲁士蓝负载血筒素纳米粒子；S40、使用所述生物仿生膜和所述普鲁士蓝负载血筒素纳米粒子制备生物仿生膜包裹普鲁士蓝负载血筒素纳米粒子；S50、对所述生物仿生膜包裹普鲁士蓝负载血筒素纳米粒子进行透明质酸修饰，得到所述血筒素靶向载药体系。A second aspect of the present invention provides a method for preparing a cytosine targeted drug-carrying system, comprising the following steps: S10, synthesizing Prussian blue nanoparticles; S20, preparing a biomimetic membrane; S30, using the Prussian blue nanoparticles and Cytolin is used to prepare Prussian blue-loaded cytolin nanoparticles; S40, use the biomimetic membrane and the Prussian blue-loaded cytolin nanoparticles to prepare a biomimetic membrane to encapsulate the Prussian blue-loaded cytolin nanoparticles; S50, to all The biomimetic membrane wraps the Prussian blue-loaded cytosine nanoparticles for hyaluronic acid modification to obtain the cytosine targeted drug-loading system.
根据本发明，所述生物仿生膜的制备方法包括如下步骤：S21、将大鼠红细胞溶血后，超声破碎，过滤，得到红细胞膜片段；S22、将类风湿关节炎成纤维样滑膜细胞低渗裂解后，超声破碎，冻融数次，离心，得到类风湿关节 炎成纤维样滑膜细胞片段；S23、将所述红细胞膜片段和所述类风湿关节炎成纤维样滑膜细胞片段等体积混合，加压过滤，离心，得到所述生物仿生膜。According to the present invention, the preparation method of the biomimetic membrane includes the following steps: S21, after hemolyzed rat erythrocytes, ultrasonically disrupted and filtered to obtain erythrocyte membrane fragments; S22, hypotonic rheumatoid arthritis fibroblast-like synovial cells After lysis, ultrasonically disrupted, freeze-thawed several times, and centrifuged to obtain rheumatoid arthritis fibroblast-like synovial cell fragments; S23, equalize the volumes of the red blood cell membrane fragments and the rheumatoid arthritis fibroblast-like synovial cell fragments Mixing, pressure filtration, and centrifugation obtain the biomimetic membrane.
根据本发明，所述普鲁士蓝负载血筒素纳米粒子的制备方法为：将血筒素和所述普鲁士蓝纳米颗粒按1：1-4的质量比混合，搅拌，透析去除未负载的血筒素，得到所述普鲁士蓝负载血筒素纳米粒子。According to the present invention, the preparation method of the Prussian blue-loaded cytolin nanoparticles is as follows: mixing the cytidine and the Prussian blue nanoparticles in a mass ratio of 1:1-4, stirring, and dialysis to remove the unloaded cytidine to obtain the Prussian blue-loaded cytoplasmin nanoparticles.
根据本发明，所述生物仿生膜包裹普鲁士蓝负载血筒素纳米粒子的制备方法为：将所述生物仿生膜和所述普鲁士蓝负载血筒素纳米粒子等体积混合，搅拌，使得所述生物仿生膜充分包裹所述普鲁士蓝负载血筒素纳米粒子，离心取沉淀，重悬，得到所述生物仿生膜包裹普鲁士蓝负载血筒素纳米粒子。According to the present invention, the preparation method of the biomimetic membrane encapsulating the Prussian blue-loaded cytonin nanoparticles is as follows: mixing the biomimetic membrane and the Prussian blue-loaded cytonin nanoparticles in equal volumes, and stirring, so that the biological The biomimetic membrane fully wraps the Prussian blue-loaded cytonin nanoparticles, centrifuged to collect the precipitate, and resuspended, to obtain the biomimetic membrane-wrapped Prussian blue-loaded cytonin nanoparticles.
根据本发明，所述透明质酸修饰的方法包括如下步骤：S51、将透明质酸中加入1-乙基-3-(3-二甲基氨基丙基)碳二亚胺和N-羟基琥珀酰亚胺搅拌活化；S52、在所述生物仿生膜包裹普鲁士蓝负载血筒素纳米粒子中加入活化后的透明质酸和磷脂-聚乙二醇-氨基，搅拌；S53、使用透析袋透析，得到所述血筒素靶向载药体系。According to the present invention, the method for modifying hyaluronic acid comprises the following steps: S51, adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinic acid to hyaluronic acid The imide is stirred and activated; S52, activated hyaluronic acid and phospholipid-polyethylene glycol-amino group are added to the biomimetic membrane-wrapped Prussian blue-loaded cytolin nanoparticles, and stirred; S53, dialysis using a dialysis bag, The hemocystin targeted drug-loading system is obtained.
本发明第三方面提供了血筒素靶向载药体系在制备抗类风湿关节炎药物中的应用。The third aspect of the present invention provides the application of the cytosine targeted drug-carrying system in the preparation of an anti-rheumatoid arthritis drug.
与现有技术相比，本发明的血筒素靶向载药体系能够靶向聚集到病变的RAFLS细胞中，提高病变部位的血筒素浓度，增强治疗效果。同时，还可以降低血液中和身体其他部位的血筒素浓度，减轻血筒素引起的不良反应。本发明的血筒素靶向载药体系的制备方法，提供了一种血筒素靶向载药体系的制备手段，能够实现对类风湿关节炎的靶向治疗。利用本发明的血筒素靶向载药体系，可以制成多种合适的药物剂型，在对类风湿关节炎进行治疗的过程中使用。Compared with the prior art, the cytosine targeted drug-loading system of the present invention can target and accumulate into the diseased RAFLS cells, increase the cytosine concentration in the diseased part, and enhance the therapeutic effect. At the same time, it can also reduce the concentration of cytosine in the blood and other parts of the body, and reduce the adverse reactions caused by cytosine. The preparation method of the cytosine targeted drug-carrying system of the present invention provides a preparation method of the cytosine targeted drug-carrying system, which can realize the targeted treatment of rheumatoid arthritis. By using the cytosine targeted drug-carrying system of the present invention, a variety of suitable pharmaceutical dosage forms can be prepared, which can be used in the treatment of rheumatoid arthritis.
附图说明Description of drawings
图1是本发明一个实施例的PB NPs扫描电镜照片；Fig. 1 is the scanning electron microscope photograph of PB NPs of an embodiment of the present invention;
图2是本发明一个实施例的PB NPs透射电镜照片；Fig. 2 is the PB NPs transmission electron microscope photograph of an embodiment of the present invention;
图3是本发明一个实施例的RAFLSM结构形成示意图；3 is a schematic diagram of the formation of a RAFLSM structure according to an embodiment of the present invention;
图4是本发明一个实施例的RBCM、RAFLSM及RFM的紫外吸收表征示意图；4 is a schematic diagram of the ultraviolet absorption characterization of RBCM, RAFLSM and RFM according to an embodiment of the present invention;
图5是本发明一个实施例得到的HA@RFM@PB@SE NPs透射电镜照片；Fig. 5 is the transmission electron microscope photograph of HA@RFM@PB@SE NPs obtained by an embodiment of the present invention;
图6是本发明一个实施例中各纳米颗粒的粒度分布曲线示意图；6 is a schematic diagram of a particle size distribution curve of each nanoparticle in an embodiment of the present invention;
图7是本发明一个实施例中各纳米颗粒的zeta电位示意图；7 is a schematic diagram of the zeta potential of each nanoparticle in an embodiment of the present invention;
图8是本发明一个实施例中各纳米颗粒的大鼠溶血示意图；8 is a schematic diagram of rat hemolysis of each nanoparticle in an embodiment of the present invention;
图9是本发明一个实施例中各纳米颗粒对RAFLS细胞的细胞活力影响示意图；9 is a schematic diagram of the effect of each nanoparticle on the cell viability of RAFLS cells in an embodiment of the present invention;
图10是本发明一个实施例中各纳米颗粒在大鼠体内分布荧光成像图；Figure 10 is a fluorescence imaging diagram of the distribution of each nanoparticle in a rat in an embodiment of the present invention;
图11是本发明一个实施例中各纳米颗粒在大鼠内脏中分布离体荧光成像图。Figure 11 is an in vitro fluorescence imaging image of the distribution of each nanoparticle in rat viscera in one embodiment of the present invention.
具体实施方式Detailed ways
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。The endpoints of ranges and any values disclosed herein are not limited to the precise ranges or values, which are to be understood to encompass values proximate to those ranges or values. For ranges of values, the endpoints of each range, the endpoints of each range and the individual point values, and the individual point values can be combined with each other to yield one or more new ranges of values that Ranges should be considered as specifically disclosed herein.
以下通过实施例对本发明进行详细说明，应当理解的是，此处所描述的 具体实施方式仅用于说明和解释本发明，本发明的保护范围并不局限于下述的具体实施方式。The present invention will be described in detail by the following examples, and it should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and the protection scope of the present invention is not limited to the following specific embodiments.
在本发明的具体实施方式中：In specific embodiments of the present invention:
所使用的实验细胞和动物：RAFLS细胞购自北京北纳创联生物技术研究院；SPF级SD大鼠购自湖南省斯莱克景达实验动物有限公司(合格证号：43004700063752)。Experimental cells and animals used: RAFLS cells were purchased from Beijing Beina Chuanglian Institute of Biotechnology; SPF SD rats were purchased from Hunan Slike Jingda Laboratory Animal Co., Ltd. (certificate number: 43004700063752).
所使用的药物和试剂：铁氰化钾(K 3[Fe(CN) 6].3H 2O)购自天津福晨化学试剂有限公司；聚(N-乙烯基吡咯烷酮)(PVP)购自上海展云化工有限公司；1×PBS(磷酸缓冲盐溶液)、0.25×PBS均购于美国Gibco公司；细胞膜蛋白提取试剂盒A液和苯甲基磺酰氟(PMSF)购自Beyotime生物技术研究所，胎牛血清(FBS)、DMEM培养基和胰酶细胞消化液购于美国Gibco公司；1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)、N-羟基琥珀酰亚胺(NHS)购自美国Sigma-Aldrich生命科学技术有限公司；磷脂-聚乙二醇-氨基(DSPE-PEG2000-NH 2)购自上海芃圣生物科技有限公司；DMSO、MTT细胞增殖及细胞毒性检测试剂盒购自上海碧云天生物技术有限公司；热灭活结核分枝杆菌H37Ra(美国Sigma Aldrich公司，20150411)。其他药物和试剂均为常规的市售产品。 Drugs and reagents used: potassium ferricyanide (K 3 [Fe(CN) 6 ].3H 2 O) was purchased from Tianjin Fuchen Chemical Reagent Co., Ltd.; poly(N-vinylpyrrolidone) (PVP) was purchased from Shanghai Zhanyun Chemical Co., Ltd.; 1×PBS (phosphate buffered saline solution) and 0.25×PBS were purchased from Gibco, USA; Cell Membrane Protein Extraction Kit Solution A and phenylmethylsulfonyl fluoride (PMSF) were purchased from Beyotime Institute of Biotechnology , fetal bovine serum (FBS), DMEM medium and trypsin cell digestion solution were purchased from Gibco, USA; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N- Hydroxysuccinimide (NHS) was purchased from Sigma-Aldrich Life Science Technology Co., Ltd. in the United States; phospholipid-polyethylene glycol-amino (DSPE-PEG2000-NH 2 ) was purchased from Shanghai Pengsheng Biotechnology Co., Ltd.; DMSO, MTT cells Proliferation and cytotoxicity detection kits were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; heat-killed Mycobacterium tuberculosis H37Ra (Sigma Aldrich, USA, 20150411). Other drugs and reagents are conventional commercially available products.
所使用的器械和仪器，扫描电子显微镜为日本電子(JEOL)のオフィシャルサイト生产，型号：JSM-6700F；超声波细胞破碎仪为南京赛飞生物科技有限公司生产，型号：Biosafer 900-92；超纯水制备系统为英国威立雅ELGA水处理技术(上海)有限公司生产，型号：FLB00003057；Zeta电位与纳米粒度分析仪为英国Malvin仪器有限公司生产，型号：Zetasizer Nano；过滤器由美国默克密理博生命科技有限公司生产；透析袋由北京索莱宝科技有限公司生 产；高速冷冻离心机(德国Eppendorf 5810R)。其他均为常见的市售产品。The instruments and instruments used, the scanning electron microscope was produced by JEOL's オフィシャルサイト, model: JSM-6700F; the ultrasonic cell disruptor was produced by Nanjing Saifei Biotechnology Co., Ltd., model: Biosafer 900-92; ultra-pure The water preparation system is produced by Veolia ELGA Water Treatment Technology (Shanghai) Co., Ltd., model: FLB00003057; the Zeta potential and nanoparticle size analyzer is produced by Malvin Instrument Co., Ltd., UK, model: Zetasizer Nano; the filter is produced by Merck Libo Life Technology Co., Ltd.; dialysis bag by Beijing Soleibao Technology Co., Ltd.; high-speed refrigerated centrifuge (Eppendorf 5810R, Germany). Others are common commercially available products.
本发明的实施方式中所用的超纯无菌水为由超纯水制备系统制备的18.2MΩ超纯水，PH为7.4。The ultrapure sterile water used in the embodiment of the present invention is 18.2MΩ ultrapure water prepared by an ultrapure water preparation system, and the pH is 7.4.
实施例1Example 1
1、普鲁士蓝纳米颗粒的合成：1. Synthesis of Prussian blue nanoparticles:
取铁氰化钾0.016g和PVP 2g，先后加入40mL 0.01M的HCl中，于室温下，搅拌溶解30min。将溶解后得到的黄色澄清溶液置于80℃油浴锅中静止反应20小时，得到粗制PB纳米材料。Take 0.016 g of potassium ferricyanide and 2 g of PVP, add them successively to 40 mL of 0.01 M HCl, and stir to dissolve for 30 min at room temperature. The yellow clear solution obtained after dissolving was placed in an oil bath at 80° C. for static reaction for 20 hours to obtain crude PB nanomaterials.
将粗制PB纳米材料分装至1.5mL EP管，在4℃环境下以13500rpm的转速离心15分钟。离心完毕后，弃去上清液，将每两管EP管中的材料合并成1管，加入1mL无菌去离子水，充分混匀后，以同样的条件离心15分钟，再将每两管EP管浓缩成1管，得到精制的PB材料。The crude PB nanomaterials were dispensed into 1.5mL EP tubes and centrifuged at 13500rpm for 15 minutes at 4°C. After centrifugation, discard the supernatant, combine the materials in each two EP tubes into one tube, add 1 mL of sterile deionized water, mix thoroughly, centrifuge under the same conditions for 15 minutes, and then add 1 mL of sterile deionized water to each tube. The EP tube was concentrated into 1 tube to obtain refined PB material.
取0.05mg/mL精制的PB材料5μL，滴加到切割好的硅片上，置于烘箱中45℃烘干，通过扫描电子显微镜拍摄，得到如图1所示的扫描电镜照片。如图1所示，得到的PB纳米颗粒为粒径为70-80nm的立方体纳米颗粒。Take 5 μL of 0.05 mg/mL refined PB material, drop it on the cut silicon wafer, place it in an oven to dry at 45°C, and photograph it with a scanning electron microscope to obtain the scanning electron microscope photo as shown in Figure 1. As shown in Figure 1, the obtained PB nanoparticles are cubic nanoparticles with a particle size of 70-80 nm.
对得到的PB纳米颗粒进行透射电镜拍摄，得到如图2所示的透射电镜照片。The obtained PB nanoparticles were photographed by a transmission electron microscope, and the transmission electron microscope photograph as shown in FIG. 2 was obtained.
由此可见，所得到的精制PB材料即为立方体PB纳米颗粒。It can be seen that the obtained refined PB materials are cubic PB nanoparticles.
2、RBCM与RAFLSM杂化生物仿生膜的制备2. Preparation of RBCM and RAFLSM hybrid biomimetic membranes
2.1、大鼠全血RBCM的提取2.1. Extraction of rat whole blood RBCM
取自大鼠的全血，用1×PBS洗涤，以3000rpm离心5min，弃去上清液，加入0.25×PBS在4℃下在中溶血1小时。将溶血的血液以12000rpm离心10min，收集RBCM。用1×PBS反复洗涤至上清液变为无色，离心收集RBCM， 重新分散在1×PBS中。将RBC膜在冰水中超声破碎30分钟。Whole blood from rats was washed with 1×PBS, centrifuged at 3000 rpm for 5 min, the supernatant was discarded, and 0.25×PBS was added for hemolysis in 1 hour at 4°C. Hemolyzed blood was centrifuged at 12000 rpm for 10 min and RBCM was collected. After repeated washing with 1×PBS until the supernatant became colorless, the RBCM was collected by centrifugation and redispersed in 1×PBS. The RBC membranes were sonicated in ice water for 30 min.
将破碎的RBCM通过0.45μm过滤器后，在通过0.22μm过滤器过滤。得到粒径小于200nm的RBCM。After passing the broken RBCM through a 0.45 μm filter, it was filtered through a 0.22 μm filter. RBCM with a particle size of less than 200 nm was obtained.
2.2、RAFLSM的提取2.2. Extraction of RAFLSM
将RAFLS细胞培养于10cm培养皿中的含10％胎牛血清(FBS)的DMEM/F-12培养基中，培养24小时，待细胞长满培养皿后，移除培养基，用1×PBS清洗，0.5％的胰酶进行消化，传代转移至15cm的大培养皿中进行培养。待细胞长满后，用刮刀收集15cm培养皿中的RAFLS细胞，用1×PBS洗涤，0.5％的胰酶将细胞全部消化，以转速800rpm，室温下离心5分钟，将获得的RAFLS细胞沉淀悬浮于0.25×PBS中低渗裂解，加入细胞膜蛋白提取试剂盒A液和PMSF(细胞膜蛋白提取试剂盒A液：PMSF＝100：1)，充分混匀后，在冰上温浴15分钟。使用超声波细胞破碎仪超声破碎5分钟，破碎功率80W，每超声破碎0.3秒，停止0.5秒。破碎后，放入-80℃中快速冷冻，反复冻融3次。将融化后的液体在4℃条件下以转速800rpm离心10分钟，取上清液进一步在4℃下以转速12000rpm离心30分钟，取沉淀物，得到细胞膜片段。RAFLS cells were cultured in DMEM/F-12 medium containing 10% fetal bovine serum (FBS) in a 10cm dish for 24 hours. Washed, digested with 0.5% trypsin, and transferred to a 15 cm large petri dish for culture. After the cells are full, collect the RAFLS cells in the 15cm culture dish with a scraper, wash with 1×PBS, digest all the cells with 0.5% trypsin, centrifuge at 800 rpm for 5 minutes at room temperature, and suspend the obtained RAFLS cell pellet. Hypotonic lysate in 0.25×PBS, add solution A of cell membrane protein extraction kit and PMSF (liquid A of cell membrane protein extraction kit: PMSF=100:1), mix well, and warm bath on ice for 15 minutes. Ultrasonic disruption was performed using an ultrasonic cell disruptor for 5 minutes, with a disruption power of 80W, each sonication was disrupted for 0.3 seconds, and the cells were stopped for 0.5 seconds. After crushing, it was quickly frozen at -80°C, and the freeze-thaw was repeated 3 times. The melted liquid was centrifuged at 800 rpm for 10 minutes at 4°C, the supernatant was further centrifuged at 12,000 rpm at 4°C for 30 minutes, and the precipitate was collected to obtain cell membrane fragments.
2.3、RBC&RAFLS生物仿生膜的制备2.3. Preparation of RBC&RAFLS biomimetic membrane
将获得的RBCM与RAFLSM等体积混合，如：取600μL的RBCM与600μL的RAFLSM混合在一起，在恒温37℃条件下超声破碎5分钟后，以800rpm的转速搅拌4小时。将混合物分别挤出1μm、400nm和200nm聚碳酸酯多孔膜以促进膜的融合。再经混合物在4℃条件下以转速21000rpm离心30分钟，收集沉淀物，即为RBC&RAFLS生物仿生膜囊泡，并将其重新悬浮于1×PBS中。Mix the obtained RBCM and RAFLSM in equal volume, for example: take 600 μL of RBCM and 600 μL of RAFLSM, mix together, sonicate for 5 minutes at a constant temperature of 37 °C, and stir at 800 rpm for 4 hours. The mixtures were extruded through 1 μm, 400 nm and 200 nm polycarbonate porous membranes, respectively, to facilitate membrane fusion. The mixture was then centrifuged at 21,000 rpm for 30 minutes at 4°C to collect the precipitate, which is RBC&RAFLS biomimetic membrane vesicles, and resuspended in 1×PBS.
如图3所示，得到RFM为由RBCM和RAFLSM交替联结而成的杂合膜。分别取10μL提取的RBCM、RAFLSM及获得的RFM，加入290μL的1×PBS，充分混匀后，用移液器加入比色皿，通过紫外分光光度计观察3种膜的紫外特征吸收峰，得到的结果如图4所示。从图4可以看出，RBCM的紫外吸收曲线在210nm和250-275nm处具有特征吸收峰，RAFLSM的紫外吸收曲线在405nm和540-560nm处具有特征吸收峰，RFM的紫外吸收曲线上具有与RBCM与RAFLSM的紫外吸收曲线叠加而成的特征吸收峰，即在波长为210nm、250-275nm、405nm和540-560nm处均有特征吸收峰。由此可知，RFM中同时具有RBCM片段和RAFLSM片段。As shown in Fig. 3, the obtained RFM is a hybrid membrane formed by alternately connecting RBCM and RAFLSM. Take 10 μL of the extracted RBCM, RAFLSM and the obtained RFM respectively, add 290 μL of 1×PBS, mix well, add to the cuvette with a pipette, observe the ultraviolet characteristic absorption peaks of the three membranes with an ultraviolet spectrophotometer, and obtain The results are shown in Figure 4. It can be seen from Figure 4 that the UV absorption curve of RBCM has characteristic absorption peaks at 210nm and 250-275nm, the UV absorption curve of RAFLSM has characteristic absorption peaks at 405nm and 540-560nm, and the UV absorption curve of RFM has the same characteristics as RBCM. The characteristic absorption peaks superimposed with the ultraviolet absorption curve of RAFLSM, that is, there are characteristic absorption peaks at the wavelengths of 210nm, 250-275nm, 405nm and 540-560nm. From this, it can be seen that the RFM has both the RBCM segment and the RAFLSM segment.
3、PB负载SE NPs的制备3. Preparation of PB-loaded SE NPs
取浓度为4mg/mL立方体PB纳米颗粒溶液366.75μL，加入浓度为500μM的SE溶液1800μL，再加入833.25μL超纯无菌水，充分混合，于25℃条件下，以800rpm转速搅拌24小时，使SE充分被负载到PB中。将混合液置于截留分子量(MWCO)为3kDa的透析袋中，在超纯水中透析12小时，去除未负载的呈游离状态的SE，得到PB负载SE NPs(PB@SE NPs)溶液。Take 366.75 μL of cubic PB nanoparticle solution with a concentration of 4 mg/mL, add 1800 μL of SE solution with a concentration of 500 μM, and then add 833.25 μL of ultrapure sterile water, mix well, and stir at 800 rpm for 24 hours at 25°C to make the solution. SE is fully loaded into PB. The mixed solution was placed in a dialysis bag with a molecular weight cut-off (MWCO) of 3 kDa, and dialyzed in ultrapure water for 12 hours to remove unloaded SE NPs in free state to obtain a PB-loaded SE NPs (PB@SE NPs) solution.
4、RFM包裹PB@SE NPs的制备4. Preparation of RFM-encapsulated PB@SE NPs
取1mL浓度为1mg/mL的RBC&RAFLS生物仿生膜(RFM)，与上一步制得的PB@SE NPs溶液等体积混合，于25℃条件下，以800rpm的转速搅拌24小时，使RFM充分包裹PB@SE NPs。将所得溶液以12000rpm的转速离心15分钟，去除上清中未包裹材料的RFM，将得到的沉淀用1×PBS重悬，得到RFM包裹PB@SE NPs(RFM@PB@SE NPs)溶液。Take 1 mL of RBC&RAFLS biomimetic membrane (RFM) with a concentration of 1 mg/mL, mix it with the PB@SE NPs solution prepared in the previous step in an equal volume, and stir at 800 rpm for 24 hours at 25 °C, so that the RFM can fully coat the PB @SE NPs. The resulting solution was centrifuged at 12,000 rpm for 15 minutes to remove the uncoated RFM in the supernatant, and the resulting pellet was resuspended with 1×PBS to obtain an RFM-coated PB@SE NPs (RFM@PB@SE NPs) solution.
5、透明质酸(HA)修饰RFM@PB@SE NPs的制备5. Preparation of hyaluronic acid (HA) modified RFM@PB@SE NPs
取100μL新鲜配制的浓度为60mg/mL的透明质酸(HA)溶液，加入1mg  EDC和0.5mg NHS，在室温下以800rpm的转速搅拌30分钟，使HA充分活化。取2mL由上一步制得的RFM@PB@SE NPs溶液，加入活化的HA和5mg DSPE-PEG-NH2，在4℃的温度下，以800rpm的转速搅拌过夜，将搅拌后的溶液置于MWCO为10kDa的透析袋中，在超纯水中透析12小时，得到透明质酸修饰RFM@PB@SE NPs(HA@RFM@PB@SE NPs)溶液，也就是本发明的血筒素靶向载药体系。Take 100 μL of freshly prepared 60 mg/mL hyaluronic acid (HA) solution, add 1 mg EDC and 0.5 mg NHS, and stir at room temperature for 30 minutes at 800 rpm to fully activate HA. Take 2 mL of the RFM@PB@SE NPs solution prepared in the previous step, add activated HA and 5 mg DSPE-PEG-NH2, stir overnight at 800 rpm at a temperature of 4 °C, and place the stirred solution in a MWCO In a 10kDa dialysis bag, dialyze in ultrapure water for 12 hours to obtain a solution of hyaluronic acid-modified RFM@PB@SE NPs (HA@RFM@PB@SE NPs), which is the cytosine targeted carrier of the present invention. drug system.
实施例2Example 2
取HA@RFM@PB@SE NPs(0.01mg/mL)溶液20μL滴于覆有碳膜的铜网上，置于红外灯下以60℃的温度烘干，使用透射电子显微镜拍摄其形状及RFM膜包裹的状态。如图5所示，HA@RFM@PB@SE NPs显示出典型的核壳结构。Take 20 μL of HA@RFM@PB@SE NPs (0.01mg/mL) solution and drop it on a copper mesh covered with carbon film, put it under an infrared lamp to dry at a temperature of 60 °C, and use a transmission electron microscope to photograph its shape and RFM film Status of the package. As shown in Figure 5, the HA@RFM@PB@SE NPs displayed a typical core-shell structure.
取PB NPs、PB@SE NPs、RFM@PB@SE NPs以及HA@RFM@PB@SE NPs各100μL置于样品比色皿中，分别使用Zeta电位与纳米粒度分析仪测量其粒径分布及Zeta电位。结构如图6、图7所示。如图6所示，PB NPs和PB@SE NPs的粒径峰值均在80nm左右，而PB@SE NPs的粒径分布更广，大粒径的颗粒更多，说明PB纳米颗粒负载SE后粒径变化不大，部分颗粒有所增大。RFM@PB@SE NPs的粒径约为140nm，显示出膜包裹后纳米颗粒的粒径明显增大，增大的粒径也与一层RFM的厚度相一致。HA@RFM@PB@SE NPs的粒径约为160nm，说明在经过HA修饰后，HA靶向材料使得纳米粒子的粒径进一步增加。如图7所示，PB NPs、PB@SE NPs、RFM@PB@SE NPs和HA@RFM@PB@SE NPs的Zeta电位分别为-16.1mV、-5.4mV、-23.6mV和-15.3mV，RFM@PB@SE NPs与HA@RFM@PB@SE NPs在包裹RFM之后Zeta电位接近，其表面电荷的绝对值升高可能与RFM囊泡包裹产生的电荷屏 蔽效应有关，而在添加靶向物质HA时同时加入了带正电荷的PEG，使得纳米粒子表面的Zeta电位相对降低，这也从另一个侧面证实了HA@RFM@PB@SE NPs的成功制备。Take 100 μL each of PB NPs, PB@SE NPs, RFM@PB@SE NPs and HA@RFM@PB@SE NPs into the sample cuvette, and use Zeta potential and nanoparticle size analyzer to measure their particle size distribution and Zeta potential. The structure is shown in Figure 6 and Figure 7. As shown in Fig. 6, the peak particle size of both PB NPs and PB@SE NPs is around 80 nm, while the particle size distribution of PB@SE NPs is wider, and there are more large-sized particles, indicating that PB nanoparticles are loaded with SE particles. The diameter did not change much, and some particles increased. The particle size of RFM@PB@SE NPs is about 140 nm, which shows that the particle size of nanoparticles increases significantly after film encapsulation, and the increased particle size is also consistent with the thickness of one layer of RFM. The particle size of HA@RFM@PB@SE NPs is about 160 nm, indicating that the HA-targeted material further increases the particle size of nanoparticles after HA modification. As shown in Fig. 7, the zeta potentials of PB NPs, PB@SE NPs, RFM@PB@SE NPs and HA@RFM@PB@SE NPs are -16.1mV, -5.4mV, -23.6mV and -15.3mV, respectively, The Zeta potential of RFM@PB@SE NPs and HA@RFM@PB@SE NPs is close to that of HA@RFM@PB@SE NPs after wrapping RFM, and the absolute increase of their surface charges may be related to the charge shielding effect produced by RFM vesicle wrapping. The addition of positively charged PEG to HA reduces the Zeta potential of the nanoparticle surface relatively, which also confirms the successful preparation of HA@RFM@PB@SE NPs from another aspect.
实施例3Example 3
取SPF级成年SD大鼠全血样，在4℃下以3000rpm离心5min，并用PBS洗涤5次，获得纯红细胞。将50μL 4％红细胞(v/v)与950μL不同浓度(浓度分别为12.5、25、50、100和200μg/mL，分散剂均为PBS)的PB NPs、PB@SE NPs、RFM@PB@SE NPs、HA@RFM@PB@SE NPs相混合，将混合液在37℃下静置4小时。阳性对照采用纯水与红细胞进行混合，混合后红细胞100％溶血。将混合液在4℃下以3000rpm的转速离心5分钟，使用UV-Vis分光光度计测定540nm处上清液的吸光度。通过公式：Hemolysis(％)＝(I/I 0)×100％计算溶血率，式中，Hemolysis为溶血率，I表示检测样品的吸光度，I 0表示阳性对照(100％溶血)的吸光度。每种样品重复进行三次，取平均值作为样品的溶血率，检测结果见图8。如图8所示，不同浓度的PB NPs、PB@SE NPs、RFM@PB@SE NPs、HA@RFM@PB@SE NPs的溶血率均在4％以下，可见，本发明的血筒素靶向载药体系具有较高的生物相容性。 Whole blood samples of SPF adult SD rats were taken, centrifuged at 3000 rpm for 5 min at 4°C, and washed 5 times with PBS to obtain pure red blood cells. 50 μL of 4% red blood cells (v/v) were mixed with 950 μL of PB NPs, PB@SE NPs, RFM@PB@SE at different concentrations (12.5, 25, 50, 100, and 200 μg/mL, respectively, dispersant in PBS). The NPs and HA@RFM@PB@SE NPs were mixed, and the mixture was allowed to stand at 37°C for 4 hours. The positive control was mixed with pure water and red blood cells, and the red blood cells were 100% hemolyzed after mixing. The mixture was centrifuged at 3000 rpm for 5 minutes at 4°C, and the absorbance of the supernatant at 540 nm was measured using a UV-Vis spectrophotometer. Calculate the hemolysis rate by the formula: Hemolysis(%)=(I/I 0 )×100%, where Hemolysis is the hemolysis rate, I represents the absorbance of the test sample, and I 0 represents the absorbance of the positive control (100% hemolysis). Each sample was repeated three times, and the average value was taken as the hemolysis rate of the sample. The detection results are shown in Figure 8. As shown in FIG. 8 , the hemolysis rates of PB NPs, PB@SE NPs, RFM@PB@SE NPs, and HA@RFM@PB@SE NPs with different concentrations were all below 4%. It has high biocompatibility to the drug-loading system.
取RAFLSs细胞，采用含100U/mL的青霉素、100μg/mL的链霉素作为双抗、接种到含10％FBS的高糖DMEM培养基中，置于含5％CO 2的培养箱中37℃培养，待细胞贴壁，生长状态良好后，2～3天传代一次。取对数生长期的RAFLS细胞，用1×PBS清洗2次，加入500μL 0.25％的胰酶细胞消化液放置于37℃培养箱中消化4min，再用1mL含10％FBS的DMEM培养基终止消化，收集细胞，置于1.5mL EP管中，900rpm的转速离心5分钟，移除上清，再加入1mL含10％FBS的DMEM培养基，制备单细胞悬浮液。再用10mL 含10％FBS的DMEM培养基制成1×10 5个/mL单细胞悬液，以每孔100μL均匀接种于96孔板中，置于含5％CO 2培养箱中37℃培养24小时。待细胞贴壁良好后，移除培养基，用1×PBS清洗1次，各孔加入100μM的含1％FBS的DMEM培养基，除Control对照组外，在阳性药组中分别加入终浓度为5μM的甲氨蝶呤和吲哚美辛，其余各孔分别加入PB终浓度为25μg/mL的PB NPs、SE终浓度为4.5μM的SE、PB@SE NPs、RFM@PB@SE NPs和HA@RFM@PB@SE NPs。其中PB NPs、PB@SE NPs、RFM@PB@SE NPs和HA@RFM@PB@SE NPs各设两个组，共设12个组，每组6个复孔。分别对PB NPs、PB@SE NPs、RFM@PB@SE NPs和HA@RFM@PB@SE NPs的一个组中的每个孔使用808nm近红外激光(1W/cm 2)照射5分钟后，将96孔板置于5％CO 2培养箱中37℃培养48小时后，弃去培养基，观察细胞生长状态。再用含10％MTT的无血清DMEM孵育4小时，弃上清液，每孔加入DMSO 100μL，在酶标仪上于492nm处检测OD值，通过公式：给药组的细胞活力＝(给药组OD值/空白对照组OD值)×100％计算细胞存活率，计算结果见图9。如图9所示，与Control组对比，PB NPs、PB NPs+Laser、SE、PB@SE NPs、PB@SE NPs+Laser、RFM@PB@SE NPs、RFM@PB@SE NPs+Laser、HA@RFM@PB@SE NPs、HA@RFM@PB@SE NPs+Laser组的RAFLS细胞活力均明显受到抑制。以Control组抑制率为0％，PB NPs、SE、PB@SE NPs、RFM@PB@SE NPs、HA@RFM@PB@SE NPs组对RAFLS细胞的抑制率分别为12.95％、27.25％、25.02％、51.58％、70.57％。使用激光照射的PB NPs+Laser、PB@SE NPs+Laser、RFM@PB@SE NPs+Laser和HA@RFM@PB@SE NPs+Laser组对RAFLS细胞的抑制率分别为28.60％、32.02％、63.68％和78.99％，与相应的未使用激光照射的给药组相比，使用 808nm的近红外激光照射后，对RAFLS细胞的抑制率更高，能够起到更好的抑制RAFLS细胞增生的效果。RFM包裹PB@SE NPs形成的RFM@PB@SE NPs和本发明的血筒素靶向载药体系HA@RFM@PB@SE NPs，对RAFLS细胞的抑制率明显高于单纯的SE和PB@SE NPs。本发明的HA@RFM@PB@SE NPs，对RAFLS细胞的抑制率达到和超过阳性药甲氨蝶呤和吲哚美辛的水平(5μM的甲氨蝶呤和吲哚美辛对RAFLS细胞的抑制率分别为70.81％与59.48％)，特别是，经过808nm的近红外激光照射后，本发明的HA@RFM@PB@SE NPs对RAFLS细胞的抑制率明显大于甲氨蝶呤及吲哚美辛，具有更好的抗类风湿关节炎作用。 Take RAFLSs cells, use 100 U/mL penicillin and 100 μg/mL streptomycin as double antibodies, inoculate into high glucose DMEM medium containing 10% FBS, and place them in an incubator containing 5% CO at 37°C Culture, and after the cells adhere to the wall and grow well, passage once every 2 to 3 days. Take the RAFLS cells in logarithmic growth phase, wash twice with 1×PBS, add 500 μL of 0.25% trypsin cell digestion solution and place them in a 37°C incubator to digest for 4 minutes, and then use 1 mL of DMEM medium containing 10% FBS to stop the digestion , the cells were collected, placed in a 1.5 mL EP tube, centrifuged at 900 rpm for 5 minutes, the supernatant was removed, and 1 mL of DMEM medium containing 10% FBS was added to prepare a single cell suspension. Then use 10 mL of DMEM medium containing 10% FBS to make 1×10 5 cells/mL single cell suspension, and evenly inoculate 100 μL per well in a 96-well plate, and place it in a 5% CO 2 incubator at 37°C. 24 hours. After the cells adhered well, the medium was removed, washed once with 1×PBS, and 100 μM DMEM medium containing 1% FBS was added to each well. Except for the Control control group, the positive drug group was added with a final concentration of 5 μM methotrexate and indomethacin, and PB NPs with a final PB concentration of 25 μg/mL, SE with a final SE concentration of 4.5 μM, PB@SE NPs, RFM@PB@SE NPs and HA were added to the other wells, respectively. @RFM@PB@SE NPs. Among them, PB NPs, PB@SE NPs, RFM@PB@SE NPs and HA@RFM@PB@SE NPs were each set up in two groups, with a total of 12 groups, each with 6 duplicate wells. After irradiating each well of a group of PB NPs, PB@SE NPs, RFM@PB@SE NPs, and HA@RFM@PB@SE NPs with 808 nm near-infrared laser (1 W/cm 2 ) for 5 min, respectively, The 96-well plate was placed in a 5% CO2 incubator at 37°C for 48 hours, the medium was discarded, and the cell growth state was observed. Incubate with serum-free DMEM containing 10% MTT for 4 hours, discard the supernatant, add 100 μL of DMSO to each well, and detect the OD value at 492 nm on a microplate reader. The OD value of the group/the OD value of the blank control group)×100% to calculate the cell survival rate, and the calculation results are shown in Figure 9. As shown in Figure 9, compared with the Control group, PB NPs, PB NPs+Laser, SE, PB@SE NPs, PB@SE NPs+Laser, RFM@PB@SE NPs, RFM@PB@SE NPs+Laser, HA The viability of RAFLS cells in the @RFM@PB@SE NPs and HA@RFM@PB@SE NPs+Laser groups was significantly inhibited. Taking the inhibition rate of Control group to be 0%, the inhibition rates of PB NPs, SE, PB@SE NPs, RFM@PB@SE NPs, and HA@RFM@PB@SE NPs groups to RAFLS cells were 12.95%, 27.25%, and 25.02, respectively. %, 51.58%, 70.57%. The inhibition rates of the PB NPs+Laser, PB@SE NPs+Laser, RFM@PB@SE NPs+Laser and HA@RFM@PB@SE NPs+Laser groups on RAFLS cells were 28.60%, 32.02%, 28.60%, 32.02%, respectively. 63.68% and 78.99%, compared with the corresponding administration group without laser irradiation, after irradiation with 808 nm near-infrared laser, the inhibition rate of RAFLS cells was higher, which could play a better effect on inhibiting RAFLS cell proliferation. . RFM@PB@SE NPs formed by encapsulating PB@SE NPs by RFM and HA@RFM@PB@SE NPs, the drug-carrying system for cylindrokinin of the present invention, have significantly higher inhibition rates on RAFLS cells than pure SE and PB@ SE NPs. The inhibition rate of HA@RFM@PB@SE NPs of the present invention on RAFLS cells reaches and exceeds the level of positive drugs methotrexate and indomethacin (5 μM methotrexate and indomethacin on RAFLS cells). The inhibition rates were 70.81% and 59.48%, respectively), especially, after 808 nm near-infrared laser irradiation, the inhibition rate of the HA@RFM@PB@SE NPs of the present invention on RAFLS cells was significantly greater than that of methotrexate and indomethacin Xin, has better anti-rheumatoid arthritis effect.
取体重为70-90g的SPF级SD大鼠分为4组，每组6只，饲养于IVC屏障系统内，保持恒温恒湿。喂养一周后，在大鼠尾根部皮下注射0.1mL含200μg热灭活结核杆菌(Mtb)的完全弗氏佐剂(CFA)建立AIA模型，免疫后18d造模成功，大鼠足趾红热肿胀。在1×PBS中分别加入N-羟基丁二酰亚胺(NHS)、1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)和ICG，形成NHS含量为1mg/mL、EDC含量为1mg/ml、ICG含量为2.5mg/mL的溶液。在室温下以800rpm的转速搅拌24小时，使用截留分子量为3.5KDa的透析袋在1×PBS中透析24小时以去除游离的ICG，得到ICG-PBS溶液。在PB@SE NPs中分别加入NHS、EDC和ICG，形成NHS含量为1mg/mL、EDC含量为1mg/ml、ICG含量为2.5mg/mL、SE含量为0.21mg/mL的溶液。在室温下以800rpm的转速搅拌24h，使用截留分子量为3.5KDa的透析袋透析24小时以去除游离的ICG，得到ICG-PB@SE NPs。依照同样的方法，分别获得SE含量为0.21mg/mL的ICG-RFM@PB@SE NPs和ICG-HA@RFM@PB@SE NPs。分别将制得的ICG-PBS、ICG-PB@SE NPs、ICG-RFM@PB@SE NPs、 ICG-HA@RFM@PB@SE NPs按1mL/Kg的量经尾静脉各注射到1只大鼠体内。使用柯达多模成像系统(Ex/Em＝740nm/820nm)分别在注射后0、6、12、24和48小时进行体内荧光成像。对于脏器分布，在注射后48小时仔细收集大鼠肿胀的足趾和主要器官(心脏，肝脏，脾脏，肺脏，肾脏)并使用柯达多模式成像系统成像。如图10、图11所示，除PBS处理的大鼠外，各大鼠均在第12-24小时内出现最强的荧光信号，此外，从第12小时起，注射HA@RFM@PB@SE NPs的大鼠体内荧光主要集中在大鼠发病的足趾关节部位，而不是均布于大鼠机体组织和各脏器中，显示出明显的靶向分布。并且，各材料在大鼠足趾部位的靶向能力为HA@RFM@PB@SE NPs>RFM@PB@SE NPs>PB@SE NPs>PBS。由此可见，本发明的血筒素靶向载药体系能够使得抗类风湿关节炎药物SE在类风湿关节炎的病变部位聚集，提高了病变部位的SE浓度，降低了身体其他组织器官中的SE浓度，从而提高了SE对类风湿关节炎的治疗作用，降低了SE对身体其他部位产生的副作用。SPF grade SD rats with a body weight of 70-90 g were taken and divided into 4 groups with 6 rats in each group, and were raised in the IVC barrier system to maintain constant temperature and humidity. After one week of feeding, 0.1 mL of complete Freund's adjuvant (CFA) containing 200 μg of heat-killed Mycobacterium tuberculosis (Mtb) was subcutaneously injected into the base of the rat's tail to establish an AIA model. The model was successfully established 18 days after immunization, and the toes of the rats were red, hot and swollen. . N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and ICG were added to 1×PBS, respectively, to form NHS with a content of 1 mg/mL, EDC content 1 mg/mL, ICG content 2.5 mg/mL solution. Stir at room temperature at 800 rpm for 24 hours, and use a dialysis bag with a molecular weight cut-off of 3.5 KDa to dialyze in 1×PBS for 24 hours to remove free ICG to obtain an ICG-PBS solution. NHS, EDC, and ICG were added to PB@SE NPs, respectively, to form solutions with NHS content of 1 mg/mL, EDC content of 1 mg/mL, ICG content of 2.5 mg/mL, and SE content of 0.21 mg/mL. After stirring at 800 rpm for 24 h at room temperature, a dialysis bag with a molecular weight cut-off of 3.5 KDa was used for 24 h to remove free ICG to obtain ICG-PB@SE NPs. Following the same method, ICG-RFM@PB@SE NPs and ICG-HA@RFM@PB@SE NPs with SE content of 0.21 mg/mL were obtained, respectively. The prepared ICG-PBS, ICG-PB@SE NPs, ICG-RFM@PB@SE NPs, and ICG-HA@RFM@PB@SE NPs were injected into one mouse via the tail vein at a volume of 1 mL/Kg, respectively. in mice. In vivo fluorescence imaging was performed at 0, 6, 12, 24 and 48 hours after injection using a Kodak Multimodal Imaging System (Ex/Em=740nm/820nm). For organ distribution, swollen toes and major organs (heart, liver, spleen, lungs, kidneys) of rats were carefully collected 48 hours after injection and imaged using the Kodak Multimodal Imaging System. As shown in Figure 10 and Figure 11, except for the PBS-treated rats, all the rats showed the strongest fluorescence signal within the 12th-24th hour. The in vivo fluorescence of SE NPs in rats was mainly concentrated in the toe joints of the rats, rather than evenly distributed in the tissues and organs of the rats, showing an obvious target distribution. Moreover, the targeting ability of each material in the rat toes was HA@RFM@PB@SE NPs>RFM@PB@SE NPs>PB@SE NPs>PBS. It can be seen that the cytonin-targeted drug-loading system of the present invention can make the anti-rheumatoid arthritis drug SE accumulate in the lesions of rheumatoid arthritis, increase the concentration of SE in the lesions, and reduce the concentration of SE in other tissues and organs of the body. SE concentration, thereby improving the therapeutic effect of SE on rheumatoid arthritis and reducing the side effects of SE on other parts of the body.
综上所述，本发明的血筒素靶向载药体系，能够与类风湿关节炎病变部位的类风湿关节炎成纤维样滑膜细胞靶向结合，提高类风湿关节炎病变部位的血筒素浓度，增强对RAFLS细胞的抑制作用，从而提高了对类风湿关节炎的治疗效果。本发明的血筒素靶向载药体系，生物相容性好，能够有效聚集在类风湿关节炎病变部位，降低身体其他部位的血筒素浓度，使用安全性高，副作用低，能够用于制备抗类风湿关节炎药物。To sum up, the targeted drug-carrying system of cytolin of the present invention can target and combine with the rheumatoid arthritis fibroblast-like synovial cells in the rheumatoid arthritis lesions, so as to improve the blood vessels in the rheumatoid arthritis lesions. The concentration of the hormone enhances the inhibitory effect on RAFLS cells, thereby improving the therapeutic effect on rheumatoid arthritis. The cytosine targeted drug-carrying system of the present invention has good biocompatibility, can effectively accumulate in the rheumatoid arthritis lesions, reduces the cytosine concentration in other parts of the body, has high safety in use, and has low side effects, and can be used for Preparation of anti-rheumatoid arthritis drugs.
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。The preferred embodiments of the present invention have been described above in detail, however, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, a variety of simple modifications can be made to the technical solutions of the present invention, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed in the present invention. All belong to the protection scope of the present invention.

Claims (10)

  1. 一种血筒素靶向载药体系，其特征在于，包括血筒素、普鲁士蓝纳米颗粒、生物仿生膜包裹层和透明质酸修饰层。A targeting drug-carrying system for cytosine is characterized by comprising cytosine, Prussian blue nanoparticles, a biomimetic membrane coating layer and a hyaluronic acid modified layer.
  2. 根据权利要求1所述的血筒素靶向载药体系，其特征在于，所述普鲁士蓝负载层为普鲁士蓝立方体纳米颗粒、普鲁士蓝球形颗粒或者普鲁士蓝立方体纳米颗粒与普鲁士蓝球形颗粒的混合物。The cytonin-targeted drug-loading system according to claim 1, wherein the Prussian blue loading layer is Prussian blue cubic nanoparticles, Prussian blue spherical particles or a mixture of Prussian blue cubic nanoparticles and Prussian blue spherical particles .
  3. 根据权利要求1所述的血筒素靶向载药体系，其特征在于，所述生物仿生膜包裹层为红细胞膜与类风湿关节炎成纤维样滑膜细胞膜组成的融合膜。The cytosine targeted drug-loading system according to claim 1, wherein the biomimetic membrane wrapping layer is a fusion membrane composed of an erythrocyte membrane and a rheumatoid arthritis fibroblast-like synovial cell membrane.
  4. 根据权利要求3所述的血筒素靶向载药体系，其特征在于，所述红细胞膜为大鼠红细胞膜。The cytosine targeted drug-loading system according to claim 3, wherein the erythrocyte membrane is a rat erythrocyte membrane.
  5. 一种血筒素靶向载药体系的制备方法，其特征在于，包括如下步骤：S10、合成普鲁士蓝纳米颗粒；A preparation method of a cytonin targeting drug-carrying system, characterized in that, comprising the following steps: S10, synthesizing Prussian blue nanoparticles;
    S20、制备生物仿生膜；S20, preparing a biomimetic membrane;
    S30、使用所述普鲁士蓝纳米颗粒和血筒素制备普鲁士蓝负载血筒素纳米粒子；S30, using the Prussian blue nanoparticles and cytolin to prepare Prussian blue-loaded cytolin nanoparticles;
    S40、使用所述生物仿生膜和所述普鲁士蓝负载血筒素纳米粒子制备生物仿生膜包裹普鲁士蓝负载血筒素纳米粒子；S40, using the biomimetic membrane and the Prussian blue-loaded cytonin nanoparticles to prepare a biomimetic membrane to wrap the Prussian blue-loaded cytonin nanoparticles;
    S50、对所述生物仿生膜包裹普鲁士蓝负载血筒素纳米粒子进行透明质酸修饰，得到所述血筒素靶向载药体系。S50 , carrying out hyaluronic acid modification on the Prussian blue-loaded cytonin nanoparticle coated with the biomimetic membrane, to obtain the cytosin-targeted drug-loading system.
  6. 根据权利要求5所述的方法，其特征在于，所述生物仿生膜的制备方法包括如下步骤：The method according to claim 5, wherein the preparation method of the biomimetic membrane comprises the following steps:
    S21、将大鼠红细胞溶血后，超声破碎，过滤，得到红细胞膜片段；S21, after hemolyzed rat erythrocytes, ultrasonically disrupted and filtered to obtain erythrocyte membrane fragments;
    S22、将类风湿关节炎成纤维样滑膜细胞低渗裂解后，超声破碎，冻融数次，离心，得到类风湿关节炎成纤维样滑膜细胞片段；S22. After hypotonic lysis of rheumatoid arthritis fibroblast-like synoviocytes, ultrasonically disrupted, freeze-thawed several times, and centrifuged to obtain rheumatoid arthritis fibroblast-like synovial cell fragments;
    S23、将所述红细胞膜片段和所述类风湿关节炎成纤维样滑膜细胞片段等体积混合，加压过滤，离心，得到所述生物仿生膜。S23. Mix equal volumes of the red blood cell membrane fragments and the rheumatoid arthritis fibroblast-like synovial cell fragments, filter under pressure, and centrifuge to obtain the biomimetic membrane.
  7. 根据权利要求5所述的方法，其特征在于，所述普鲁士蓝负载血筒素纳米粒子的制备方法为：将血筒素和所述普鲁士蓝纳米颗粒按1:1-4的质量比混合，搅拌，透析去除未负载的血筒素，得到所述普鲁士蓝负载血筒素纳米粒子。The method according to claim 5, characterized in that, the preparation method of the Prussian blue-loaded cytonin nanoparticles is: mixing the cytonin and the Prussian blue nanoparticles in a mass ratio of 1:1-4, With stirring, the unloaded cytidine was removed by dialysis to obtain the Prussian blue-loaded cytolin nanoparticles.
  8. 根据权利要求5所述的方法，其特征在于，所述生物仿生膜包裹普鲁士蓝负载血筒素纳米粒子的制备方法为：将所述生物仿生膜和所述普鲁士蓝负载血筒素纳米粒子等体积混合，搅拌，使得所述生物仿生膜充分包裹所述普鲁士蓝负载血筒素纳米粒子，离心取沉淀，重悬，得到所述生物仿生膜包裹普鲁士蓝负载血筒素纳米粒子。The method according to claim 5, wherein the preparation method of the biomimetic membrane wrapping the Prussian blue-loaded cytonin nanoparticles is as follows: the biomimetic membrane and the Prussian blue-loaded cytosine nanoparticles are mixed together, etc. Volume mixing, stirring, so that the biomimetic membrane fully wraps the Prussian blue-loaded cytonin nanoparticles, centrifuged to take the precipitate, and resuspended to obtain the biomimetic membrane-wrapped Prussian blue-loaded cytonin nanoparticles.
  9. 根据权利要求5所述的方法，其特征在于，所述透明质酸修饰的方法包括如下步骤：The method according to claim 5, wherein the method for modifying hyaluronic acid comprises the steps:
    S51、将透明质酸中加入1-乙基-3-(3-二甲基氨基丙基)碳二亚胺和N- 羟基琥珀酰亚胺搅拌活化；S51, adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to hyaluronic acid with stirring and activation;
    S52、在所述生物仿生膜包裹普鲁士蓝负载血筒素纳米粒子中加入活化后的透明质酸和磷脂-聚乙二醇-氨基，搅拌；S52, adding activated hyaluronic acid and phospholipid-polyethylene glycol-amino to the Prussian blue-loaded cytonin nanoparticles wrapped in the biomimetic membrane, and stirring;
    S53、使用透析袋透析，得到所述血筒素靶向载药体系。S53, using a dialysis bag for dialysis to obtain the cytosine targeted drug-carrying system.
  10. 血筒素靶向载药体系在制备抗类风湿关节炎药物中的应用。The application of cytoplasmin targeting drug-loading system in the preparation of anti-rheumatoid arthritis drugs.
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