CN115944616A - Application of etamsylate and nano preparation thereof in preparation of medicine for treating spinal cord injury - Google Patents
Application of etamsylate and nano preparation thereof in preparation of medicine for treating spinal cord injury Download PDFInfo
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- CN115944616A CN115944616A CN202211644237.4A CN202211644237A CN115944616A CN 115944616 A CN115944616 A CN 115944616A CN 202211644237 A CN202211644237 A CN 202211644237A CN 115944616 A CN115944616 A CN 115944616A
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- etamsylate
- spinal cord
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- cord injury
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Images
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to application of etamsylate and a nano preparation thereof in preparation of a medicine for treating spinal cord injury. The invention provides application of etamsylate in preparation of a medicine for treating spinal cord injury. The embodiment of the invention shows that: the invention adopts a rat spinal cord contusion model, selects the etamsylate as an intervention drug, selects the etamsylate as a hemostasis intervention drug, does not observe obvious thrombosis, and prompts the etamsylate to be a potential drug for clinically treating early bleeding caused by spinal cord injury. The phenasulfoethylamine is injected into tail veins at different time points immediately after spinal cord contusion and after injury respectively, and the phenasulfoethylamine is found to be capable of effectively reducing the area of an injury area and the number of necrotic cells, increasing neuron survival in a parainjury area, relieving astrocyte and microglia/macrophage activation, and promoting motor function recovery of rats after spinal cord injury.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of etamsylate and a nano preparation thereof in preparation of a medicine for treating spinal cord injury.
Background
Spinal cord injury is the most common nervous system injury, with the continuous development of the building industry, the transportation industry, the sports industry and the like, the population aging is overall aggravated, the incidence of the injuries is increased year by year, the trend of the aging is more and more obvious, the serious threat is brought to the life health of patients, and the economic burden of bearing great load is caused to the family society.
Among them, bleeding after spinal cord injury is an important pathological phenomenon leading to neuronal death, aggravation of secondary injury and loss of function. According to research, within 12 hours after spinal cord injury of rats, non-ruptured red blood cells exist in the local injured area, which shows that the duration of blood brain barrier bleeding after injury is long, and a large amount of harmful substances released after rupture can cause secondary injury of the spinal cord. Thus, early hemostasis is beneficial to reducing secondary injury. However, the body is in a high coagulation state after the trauma, and excessive hemostasis can cause deep vein embolism events, and even endanger life in severe cases.
Disclosure of Invention
The invention aims to provide the application of the etamsylate and the nano preparation thereof in preparing the medicine for treating spinal cord injury, the etamsylate and the nano preparation thereof provided by the invention have no obvious thrombosis when being used for spinal nerve hemostasis, can obviously promote motor function recovery after spinal cord injury when being used for treating the spinal cord injury, have no obvious thrombosis, and are potential medicines for clinically treating early bleeding of the spinal cord injury.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides application of etamsylate in preparing a medicine for treating spinal cord injury.
Preferably, the treatment of spinal cord injury is hemostasis of spinal nerves.
Preferably, the effective dosage of the etamsylate in the medicine is 0.1g/kg.
Preferably, the etamsylate is an etamsylate nano-preparation.
Preferably, the particle size of the etamsylate nanometer preparation is 60-100 nm.
Preferably, the etamsylate nanometer preparation comprises etamsylate and a drug carrier.
Preferably, the drug carrier comprises one or more of polyester, chitosan, gelatin, photopolymerizable hydrogel, liposomes, starch and sodium alginate.
Preferably, the polyester comprises one or more of polycaprolactone, glycolide-L-lactide-caprolactone ternary random copolymer, poly-DL-lactic acid-polyethylene glycol copolymer, poly-L-lactide and polylactic acid-glycolic acid copolymer.
Preferably, the photopolymerizable hydrogel includes one or more of methacrylated chitosan, methacrylated gelatin, methacrylated silk fibroin, methacrylated hyaluronic acid, methacrylated alginic acid, and methacrylated dextran;
the liposome is prepared from phospholipid, cholesterol and alpha-tocopherol.
The invention provides application of etamsylate in preparation of a medicine for treating spinal cord injury. The embodiment of the invention shows that: the invention adopts rat spinal cord contusion and hemisection models respectively, selects the etamsylate as an intervention drug, selects the etamsylate as a hemostasis intervention drug, does not observe obvious thrombosis, and prompts the etamsylate to be a potential drug for clinically treating early bleeding caused by spinal cord injury. The phenasulfoethylamine is injected into tail veins at different time points immediately after spinal cord contusion and after spinal cord contusion respectively, and the phenasulfoethylamine is found to effectively reduce the area of a damaged area and the number of necrotic cells, increase the survival of neurons in a damaged area, relieve the activation of astrocytes and microglia/macrophages, and promote the recovery of motor function of rats after spinal cord injury.
Further, in the invention, the etamsylate is an etamsylate nanometer preparation. The embodiment of the invention shows that: compared with a control group and a common preparation group sold in the market of etamsylate, the nano preparation hemostatic provided by the invention can obviously reduce the transcription levels of proinflammatory immune factors TNF alpha, iNOS and IL-12 after rat spinal cord injury; can obviously increase the transcription levels of the inflammatory-inhibiting immune factors Arginase1, ym1, IL-4 and IL-10 after the spinal cord injury of the rat; can increase the neuron survival in the injury area after the spinal cord injury of the rat and the residual nerve fiber quantity around and in the injury area after the spinal cord injury of the rat. The results indicate that the nano-preparation of the etamsylate provided by the invention not only has a hemostatic effect and can relieve the secondary injury of the spinal cord, but also can damage a local immune microenvironment after the nano-preparation reaches the local part of the injured tissue, and provide a good living environment for the survival of the peripheral neurons in the injured area, so that the survival of the peripheral neurons in the injured area is promoted, the number of nerve fibers is increased, and the recovery of the motor function after the spinal cord injury is promoted. Therefore, compared with a control group and a commercial common preparation of the etamsylate (national drug group, vol. Pharmaceutical Co., ltd., 10, each specification of 2mL.
Drawings
FIG. 1 is a graph of BBB score changes at different time points after spinal cord injury in rats for different dosage forms of etamsylate;
FIG. 2 is a graph showing the effect of different etamsylate dosage forms on the transcriptional levels of the pro-inflammatory immune factors TNF α, iNOS, IL-12 following spinal cord injury in rats;
FIG. 3 is a graph showing the effect of different etamsylate dosage forms on the transcriptional levels of the anti-inflammatory immune factors Arginase1, ym1, IL-4, IL-10 following spinal cord injury in rats;
FIG. 4 is a graph of the effect of different etamsylate formulations on the survival of neurons in the parainjury area following spinal cord injury in rats;
FIG. 5 is a graph showing the effect of different etamsylate formulations on the number of residual nerve fibers around and in the center of the injured area after spinal cord injury in rats.
Detailed Description
The invention provides application of etamsylate in preparation of a medicine for treating spinal cord injury.
In the present invention, all the preparation starting materials/components are commercially available products well known to those skilled in the art unless otherwise specified.
In the present invention, the treatment of spinal cord injury is preferably hemostasis of spinal cord nerves.
In the invention, the medicine for treating bleeding caused by spinal cord injury is preferably a medicine for treating bleeding at the early stage of spinal cord injury.
In the present invention, the administration time of the drug for treating bleeding at the early stage of spinal cord injury is preferably within 24 hours, and more preferably within 10 hours.
In the invention, the effective dosage of the etamsylate in the spinal cord nerve hemostasis medicine or the medicine for treating spinal cord injury is preferably 0.1g/kg.
In the present invention, the effective amount is a proportional relationship between the mass of the etamsylate and the weight of the patient.
In the invention, the etamsylate is preferably an etamsylate nanoformulation.
In the invention, the particle size of the nano-preparation of etamsylate is preferably 60-100 nm.
In the present invention, the nano-formulation of etamsylate preferably comprises etamsylate and a pharmaceutical carrier.
In the present invention, the drug carrier preferably comprises one or more of polyester, chitosan, gelatin, photopolymerizable hydrogel, liposome, starch and sodium alginate.
In the present invention, the polyester preferably includes one or more of Polycaprolactone (PCL), glycolide-L-lactide-caprolactone terpolymer (PGLC), poly-DL lactic acid-polyethylene glycol copolymer (PELA), poly-L-lactide (PLLA) and polylactic acid-glycolic acid copolymer (PLGA).
The photopolymerizable hydrogel preferably includes one or more of methacrylated chitosan, methacrylated gelatin, methacrylated silk fibroin, methacrylated hyaluronic acid, methacrylated alginic acid, and methacrylated dextran.
In the present invention, the liposome is prepared from phospholipid, cholesterol and alpha-tocopherol.
In the present invention, the starch is preferably soluble starch.
In the present invention, the preparation method of the etamsylate nano-formulation is preferably selected according to the kind of the pharmaceutical carrier.
In the invention, when the drug carrier is polyester, the preparation method of the etamsylate nano preparation preferably comprises a filtration method, a homogeneous emulsification method, an ultrasonic emulsification method, a membrane emulsification method or a spray drying method.
In the present invention, when the drug carrier is chitosan and/or gelatin, the preparation method of the nano-preparation of etamsylate preferably comprises a phacoemulsification method or a crosslinking method.
In the invention, when the drug carrier is a photo-polymerization hydrogel, the preparation method of the etamsylate nanometer preparation preferably comprises a photo-curing method or a microfluidic method
In the present invention, when the drug carrier is a liposome, the preparation method of the etamsylate nano-formulation preferably includes a thin film dispersion method.
In the invention, when the carrier to be removed is starch, the preparation method of the etamsylate nano preparation preferably comprises a reverse microemulsion method.
In the invention, when the drug carrier is sodium alginate, the preparation method of the nano-phenolsulfoethylamine preparation preferably comprises a micro-fluidic method or an emulsifying ion crosslinking method.
In the present invention, the filtration method preferably comprises the steps of:
dissolving the polyester in an organic solvent to obtain a polyester solution;
mixing the polyester solution, the etamsylate, a surfactant and water to obtain a dispersion liquid;
removing the organic solvent from the dispersion to obtain an aqueous suspension;
and filtering the aqueous suspension membrane, and centrifuging to obtain the etamsylate nano preparation.
In the present invention, the organic solvent includes tetrahydrofuran and ethanol. The volume ratio of tetrahydrofuran to ethanol is preferably 3. The temperature of the mixing is preferably 25 to 40 ℃. The surfactant is preferably tween, and the volume ratio of the surfactant to the water is preferably 1. The method for removing the organic solvent is preferably rotary evaporation, and the temperature of the rotary evaporation is preferably room temperature. The pore size of the filter membrane used in the membrane filtration is preferably 0.45 μm. The time for the centrifugation is preferably 15min. The solid-phase product is obtained through centrifugation, and the solid-phase product is preferably subjected to post-treatment to obtain the etamsylate nano preparation. In the present invention, the post-treatment preferably comprises the steps of: and (3) washing and drying the solid-phase product in sequence. In the present invention, the number of the water washing is preferably 2, and the water washing is preferably distilled water washing. The drying is preferably freeze-drying.
In the present invention, the homogeneous emulsification method preferably comprises the steps of:
mixing the polyester and an organic solvent to obtain an oil phase;
mixing polyvinyl alcohol (PVA), etamsylate and water to obtain a water phase;
homogenizing and emulsifying the oil phase and the water phase under the condition of ice-water bath to obtain oil/water emulsion;
and removing the organic solvent from the oil/water emulsion to obtain the etamsylate nano preparation.
In the present invention, the organic solvent is preferably dichloromethane. The homogenizing emulsification is preferably carried out in a homogenizing emulsifier. The temperature for removing the organic solvent is preferably room temperature. The organic solvent is removed to obtain a solid-phase product, and the solid-phase product is preferably subjected to post-treatment to obtain the etamsylate nano preparation. In the present invention, the post-treatment preferably comprises the steps of: and washing and drying the solid-phase product in sequence. In the present invention, the number of the water washing is preferably 2, the water washing is preferably ionic water washing, and centrifugal separation is preferably used between the two water washing. The drying is preferably freeze-drying.
In the present invention, the phacoemulsification method preferably includes the steps of:
mixing the polyester and an organic solvent to obtain an oil phase;
mixing polyvinyl alcohol (PVA), etamsylate and water to obtain a water phase;
under the condition of ice-water bath, ultrasonically emulsifying the oil phase and the water phase to obtain oil/water emulsion;
and removing the organic solvent from the oil/water emulsion to obtain the etamsylate nano preparation.
In the present invention, the organic solvent is preferably dichloromethane. The method for removing the organic solvent is preferably a solvent evaporation method. The organic solvent is removed to obtain a solid-phase product, and the solid-phase product is preferably subjected to post-treatment to obtain the etamsylate nano preparation. In the present invention, the post-treatment preferably comprises the steps of: and washing and drying the solid-phase product in sequence. In the present invention, the number of the water washing is preferably 2, the water washing is preferably ionic water washing, and centrifugal separation is preferably used between the two water washing. The drying is preferably freeze-drying.
In the present invention, the membrane emulsification method preferably includes method one or method two.
In the present invention, the first method preferably comprises the steps of:
mixing the polyester with an organic solvent to obtain an oil phase;
mixing etamsylate and water to obtain an internal water phase;
under the condition of ice-water bath, ultrasonically emulsifying the oil phase and the internal water phase to obtain a water-in-oil primary emulsion;
adding the water-in-oil primary emulsion into a polyvinyl alcohol aqueous solution, and mixing to obtain a pre-composite emulsion;
under the condition of nitrogen pressure, performing membrane emulsification on the pre-compound emulsion to obtain compound emulsion;
and removing the organic solvent from the double emulsion, and then carrying out solid-liquid separation to obtain the etamsylate nanometer preparation.
In the present invention, the organic solvent is preferably dichloromethane. The phacoemulsification is preferably carried out in an ultrasonic cell disruptor. The mixing of the water-in-oil primary emulsion into the polyvinyl alcohol aqueous solution is preferably carried out under the condition of mechanical stirring, and the time of the mechanical stirring is preferably 2-5 min. The membrane emulsification is preferably: pouring the pre-emulsion into a storage tank of a rapid membrane emulsification device, repeatedly pressing the pre-emulsion through an SPG membrane with the aperture of 0.5-1.2 mu m under the pressure of nitrogen until the emulsion with uniform particle size is obtained. The temperature for removing the organic solvent is preferably room temperature. The organic solvent is removed to obtain a solid-phase product, and the solid-phase product is preferably subjected to post-treatment to obtain the etamsylate nano preparation. In the present invention, the post-treatment preferably comprises the steps of: and washing and drying the solid-phase product in sequence. In the present invention, the water washing is preferably distilled water washing, and centrifugal separation is preferably used between the two water washing. The drying is preferably liquid nitrogen freeze drying.
In the present invention, the second method preferably comprises the steps of:
mixing aqueous solution of chitosan and/or gelatin and acetic acid with etamsylate to obtain water phase;
mixing the paraffin, petroleum ether and an emulsifier to obtain an oil phase;
mixing the water phase and the oil phase to obtain a pre-emulsion;
performing membrane emulsification on the pre-emulsion under the condition of nitrogen pressure to obtain water-oil emulsion;
and mixing and crosslinking the water-oil emulsion and the crosslinking agent organic solution to obtain the etamsylate nano preparation.
In the present invention, the content of the aqueous solution of acetic acid is preferably 1% by mass. The membrane emulsification is preferably: pouring the pre-emulsion into a storage tank of a rapid membrane emulsification device, and repeatedly pressing the pre-emulsion through an SPG membrane with the aperture of 0.5-1.2 mu m under the pressure of nitrogen until the emulsion with uniform particle size is obtained. The cross-linking agent is preferably a glutaraldehyde-saturated toluene solution. In the invention, the crosslinking reaction liquid is obtained after mixing and crosslinking, and the invention preferably carries out post-treatment on the crosslinking reaction liquid to obtain the etamsylate nano preparation. In the present invention, the post-treatment preferably comprises the steps of: centrifuging the crosslinking reaction solution to obtain a solid-phase product; and washing and drying the solid-phase product in sequence. In the present invention, the washing is preferably carried out by petroleum ether washing, acetone washing and ethanol washing in this order. The drying is preferably freeze drying.
In the present invention, the spray drying method preferably comprises the steps of:
mixing the polyester, the etamsylate and the organic solvent to obtain a suspension;
and carrying out spray drying on the suspension to obtain the etamsylate nanometer preparation.
In the present invention, the organic solvent is preferably dichloromethane. The superspray drying is preferably carried out in a nanospray dryer. The inlet temperature of the super-spray drying is preferably 60 ℃, and the pore diameter of the spray head cap of the super-spray drying is preferably 0.5-1.5 μm. The invention preferably stores the etamsylate nanometer preparation in a freezing way.
In the present invention, the crosslinking method preferably includes the steps of:
mixing chitosan and/or gelatin, etamsylate and water to obtain a mixed solution;
mixing paraffin and an emulsifier to obtain an emulsified solution;
injecting the emulsified solution into the mixed solution to obtain emulsion;
mixing and solidifying the emulsion and the cross-linking agent, and adding an organic solvent to obtain a nanoparticle dispersion liquid;
and centrifuging the nanoparticle dispersion liquid to obtain the etamsylate nano preparation.
In the present invention, when the chitosan raw material is used in the crosslinking method, the mixed raw material preferably further includes acetic acid. The temperature at which the chitosan and/or gelatin, the etamsylate and the water are mixed is preferably 70 ℃. The emulsifier is preferably Span80. The temperature at which the paraffin wax and the emulsifier are mixed is preferably 55 ℃. The mixing of the paraffin wax and the emulsifier is preferably carried out under mechanical stirring. The present invention preferably injects the emulsified solution into the mixed solution using a syringe. After the injection, the present invention preferably continues stirring for 15min to obtain an emulsion. In the present invention, the crosslinking agent is preferably an aqueous glutaraldehyde solution. The mass percentage content of the glutaraldehyde water solution is preferably 12.5%. The crosslinking agent is preferably added in two portions. The organic solvent is preferably acetone. The time for the centrifugation is preferably 15min. The solid-phase product is obtained through centrifugation, and the solid-phase product is preferably subjected to post-treatment to obtain the etamsylate nano preparation. In the present invention, the post-treatment preferably comprises the steps of: and washing and drying the solid-phase product in sequence. In the present invention, the washing is preferably rinsing with acetone and isopropyl alcohol in this order. The drying is preferably freeze drying.
In the present invention, the photo-curing method preferably includes the steps of:
first mixing the photo-polymerization hydrogel, etamsylate, lithium acylphosphinate and water to obtain a pre-mixed solution;
secondly, mixing the edible oil and the paraffin oil to obtain mixed oil;
thirdly mixing the premixed solution and the mixed oil to obtain a mixed solution;
and curing the mixed solution under the ultraviolet irradiation condition to obtain the etamsylate nano preparation.
In the present invention, the temperature of the first mixing is preferably 40 ℃, and the first mixing is preferably performed under water bath conditions. The temperature of the second mixing is preferably 60 ℃, and the second mixing is preferably performed under early-stirring conditions. The third mixing is preferably performed by dropping the premixed solution into the mixed oil. The curing is preferably carried out under stirring, and the curing time is preferably 5min. In the invention, the solidification is carried out to obtain a solidified product, and the solidified product is preferably subjected to post-treatment to obtain the etamsylate nano preparation. In the present invention, the post-treatment preferably comprises the steps of: and standing the solidified product, and then washing and drying the solidified product in sequence. In the present invention, the washing is preferably performed by sequentially washing with pre-cooled dioxane, acetone and ethanol. The drying is preferably oven drying, the temperature of the drying preferably being 60 ℃.
In the present invention, the microfluidic method preferably includes method three or method four.
In the present invention, the third method preferably comprises the following steps:
mixing the photo-polymerization hydrogel, etamsylate, a buffer solution and a photoinitiator to obtain an internal phase solution;
mixing mineral oil and a surfactant to obtain a continuous phase solution;
injecting the inward solution and the continuous phase solution into two inlets of the microfluidic device to be mixed to obtain shear microdroplets;
and under the condition of ultraviolet irradiation, carrying out photo-crosslinking curing on the sheared microdroplets to obtain the etamsylate nano preparation.
In the present invention, the internal phase solution preferably contains the photoinitiator in an amount of 0.5% by mass. The surfactant is preferably Span80. The mass ratio of the mineral oil to the surfactant is preferably 1. In the invention, the solid gel nanoparticles are obtained through photo-crosslinking and curing, and the solid gel nanoparticles are preferably subjected to post-treatment to obtain the etamsylate nano preparation. In the present invention, the post-treatment preferably comprises the steps of: and washing and drying the solid gel nanoparticles in sequence. In the invention, the washing is preferably carried out by sequentially using acetone and 75% by mass of an ethanol aqueous solution. The drying is preferably freeze-drying.
In the present invention, the method four preferably comprises the steps of:
mixing the etamsylate, the buffer solution and the sodium alginate to obtain an internal phase solution;
mixing an ionic cross-linking agent and water to obtain an external water phase;
and injecting the inward solution, the continuous phase and the external water phase into three inlets of a microfluidic device, mixing, shearing and crosslinking to obtain the etamsylate nano preparation.
In the present invention, the buffer solution is preferably a PBS solution. The continuous phase is preferably mineral oil. The ionic crosslinking agent is preferably CaCl 2 And ZnCl 2 (ii) a In the external water phase, caCl 2 Is preferably 5%, and the ZnCl 2 The mass percentage of (b) is preferably 10%. The present invention preferably employs a syringe pump to inject the internal solution, continuous phase and external aqueous phase into the microfluidic device. In the present invention, the mixing shear crosslinking gives a microdroplet, and the present invention preferably performs post-treatment on the microdroplet to give the etamsylateA nanometer medicinal preparation. In the present invention, the post-treatment preferably comprises the steps of: the droplets are washed and dried sequentially. In the invention, the washing is preferably carried out by sequentially using acetone and 75% by mass of an ethanol aqueous solution. The drying is preferably freeze-drying.
In the present invention, the preparation method of the thin film dispersion method preferably includes the steps of:
first mixing the phospholipids, cholesterol, alpha-tocopherol and a first organic solvent to obtain a lipid solution;
secondly, mixing the lipoid solution, the etamsylate and a second organic solvent to obtain a mixed solution;
removing the organic solvent from the mixed solution to obtain a lipoid film;
hydrating the lipoid film by a hydration medium dissolved with a freeze-drying protective agent to obtain a coarse suspension;
and carrying out ultrasonic treatment on the crude suspension to obtain the etamsylate nanometer preparation.
In the present invention, the first organic solvent is preferably chloroform. The second organic solvent is preferably methanol. The temperature of the second mixing is preferably 40 ℃, and the second mixing is preferably carried out under water bath conditions. The method for removing the organic solvent is preferably distillation under reduced pressure. The time of the ultrasonic treatment is preferably 2 to 10min. The invention preferably stores the nano-preparation of the etamsylate in a refrigerator at 4 ℃.
In the present invention, the reverse microemulsion method preferably includes the steps of:
firstly mixing starch, naOH, etamsylate and water to obtain an aqueous phase solution;
secondly, mixing the soybean oil and the surfactant to obtain an oil phase solution;
thirdly mixing the water phase solution and the oil phase solution to obtain a dispersed emulsion;
and mixing the dispersed emulsion with epoxy chloropropane, and performing centrifugal separation to obtain the etamsylate nano preparation.
In the present invention, the first mixing is preferably performed under stirring, and after the first mixing, the aqueous phase solution is preferably activated at room temperature for 30min. The temperature of the second mixing is preferably 60 ℃. The temperature of the first mixing is preferably 40 ℃, and the third mixing is preferably: the aqueous phase solution was slowly added to the oil phase solution. In the invention, the centrifugal separation is carried out to obtain a solid-phase product, and the solid-phase product is preferably subjected to post-treatment to obtain the etamsylate nano preparation. In the present invention, the post-treatment preferably comprises the steps of: and washing and drying the solid-phase product in sequence. In the present invention, the washing is preferably repeated with ethyl acetate washing, ethanol and acetone washing. The drying is preferably carried out in a drying oven. The etamsylate nanometer preparation is preferably frozen for storage.
In the present invention, the preparation method of the emulsifying ion crosslinking method preferably includes the steps of:
mixing the etamsylate solution and the sodium alginate solution to obtain a water phase;
mixing the corn oil and a surfactant to obtain an oil phase;
dropwise adding the water phase into the oil phase to obtain a water/oil emulsion;
and mixing the water/oil emulsion and an ionic cross-linking agent for cross-linking and curing to obtain the etamsylate nano preparation.
In the present invention, the etamsylate solution is preferably etamsylate PBS solution. The mass percentage content of the sodium alginate solution is preferably 1.5%. The volume ratio of the etamsylate solution to the sodium alginate solution is preferably 3. The surfactant preferably comprises span-85 and tween-85; the volume ratio of the corn oil to the span-85 to the tween-85 is preferably 100. The dripping is preferably carried out under the condition of stirring, the rotating speed of the stirring is preferably more than or equal to 4000r/min, and the stirring time is preferably 5min. The ionic crosslinking agent is preferably CaCl 2 And ZnCl 2 The mixed solution of (1); said CaCl 2 And ZnCl 2 In the mixed solution of (2), caCl 2 Is preferably 5%, and the ZnCl 2 Is preferably contained in percentage by massIs 10%. The crosslinking curing is preferably carried out under stirring, and the time for crosslinking curing is preferably 15min. In the invention, the crosslinking and curing are carried out to obtain a curing reaction liquid, and the invention preferably carries out post-treatment on the curing reaction liquid to obtain the etamsylate nano preparation. In the present invention, the post-treatment preferably includes: centrifuging the solidification reaction liquid to obtain nanoparticles; and washing and drying the nanoparticles in sequence. In the present invention, the washing is preferably 3 times with sterilized water for injection. The drying is preferably freeze drying.
In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Dissolving 0.5g of Polyester (PCL) in 10mL of tetrahydrofuran, and adding ethanol, wherein the volume ratio of the tetrahydrofuran to the ethanol is 3/2, so as to obtain a polymer solution; then 0.1g of etamsylate was added to the polymer solution to obtain a homogeneous mixed solution. The mixed solution was added dropwise to 10mL of Tween and 60mL of an aqueous solution under mild stirring at 40 ℃ to form a dispersion. After tetrahydrofuran and ethanol were completely removed from the dispersion by rotary evaporation at room temperature, the resulting aqueous suspension was filtered through a 0.45 μm filter to remove precipitates. The polyester nanoparticles loaded with etamsylate were collected by centrifugation at 8000rpm for 15 minutes and washed 2 times with distilled water. After freeze-drying, the dried polyester-supported etamsylate nanoparticles are obtained.
Example 2
Dissolving 0.5g of Polyester (PCL) in 10mL of dichloromethane to serve as an oil phase, dispersing the oil phase in the water phase by using 10mL of aqueous solution containing 0.1% of PVA by mass and 0.1% of etamsylate by mass as a water phase under the ice bath condition by using a high-speed homogenizing emulsifying machine (the rotating speed is 4000 r/min) to prepare oil/water emulsion, magnetically stirring and volatilizing the oil/water emulsion at normal temperature to remove organic solvent and solidify the organic solvent into nanoparticles, centrifugally washing the nanoparticles for several times by using deionized water, and freeze-drying the nanoparticles to prepare the finished product.
Example 3
Dissolving Polyester (PCL) in dichloromethane to serve as an oil phase (the mass percent of PCL in the oil phase is 1%), taking PVA and etamsylate aqueous solution as an aqueous phase (the mass percent of etamsylate in the aqueous phase is 0.1%, and the mass percent of PVA is 0.1%), dispersing 10mL of the oil phase in 10mL of the aqueous phase by ultrasonic emulsification under an ice bath condition to prepare oil/water emulsion, removing an organic solvent in emulsion droplets by a solvent volatilization method to solidify the organic solvent into microspheres, centrifugally washing the microspheres for several times by deionized water at 8000rpm, and freeze-drying the microspheres to prepare a finished product.
Example 4
Taking 10mL of a phenolsulfoethylamine aqueous solution as an internal water phase (0.1%), and dissolving Polyester (PCL) in 10mL of dichloromethane to obtain an oil phase (the mass percentage of the PCL is 1%). The internal aqueous phase was added to 10mL of the oil phase, and ultrasonic emulsification was performed using an ultrasonic cell disruptor to obtain a water-in-oil primary emulsion. Then, the primary emulsion is poured into a polyvinyl alcohol aqueous solution (the mass percentage content is 0.1 percent), and is stirred at 3000r/min for 5min under mechanical stirring for pre-composite emulsification. Subsequently, the pre-emulsion was poured into a storage tank of a rapid membrane emulsification apparatus, and the pre-emulsion was repeatedly pressed through an SPG membrane having a pore diameter of 1.2 μm with nitrogen gas until an emulsion having a uniform particle diameter was obtained. Finally, the double emulsion was stirred at room temperature overnight to completely evaporate the solvent. And (3) centrifugally collecting the solidified nanoparticles at 8000rpm, washing the nanoparticles with distilled water for several times, freezing the nanoparticles by using liquid nitrogen, and freeze-drying the nanoparticles by using a freeze dryer to obtain the drug-loaded nanoparticles with uniform particle size.
Example 5
Weighing Polyester (PCL), dissolving the Polyester (PCL) in dichloromethane (the mass percentage of the PCL is 1%), ultrasonically dispersing 0.1g of etamsylate powder in 10mL of dichloromethane solution dissolved with the PCL, and performing spray drying on the prepared suspension by adopting a nano spray dryer. The inlet temperature was set at 60 ℃ and the nozzle cap aperture was 0.5. Mu.m. Collecting the dried drug-loaded nanoparticles for freezing and storing.
Example 6
Dissolving 1g of chitosan in 10mL of acetic acid-etamsylate solution (the mass percentage of acetic acid is 1 percent, and the mass percentage of etamsylate is 0.3 percent) at 70 ℃, fully dissolving the chitosan, and stirring at room temperature to uniformly mix the solution. Then adding an emulsifier Span80 (the mass percentage of the Span80 is 1%) into 10mL of liquid paraffin; mechanically stirring at high speed of 4000r/min at constant temperature of 55 deg.C, slowly adding the chitosan mixed solution with an injector, and continuously stirring for 15min to form stable emulsion. After stirring for a further 30min at room temperature, 5mL of crosslinker (12.5% strength aqueous glutaraldehyde solution, 2.5mL each) were added in two portions and allowed to solidify. Adjusting the stirring speed to 3000r/min, and then adding acetone to dehydrate the nanoparticles. Centrifuging at 8000rpm for 15min to obtain nanoparticles. And respectively rinsing with acetone and isopropanol, and freeze-drying to obtain the chitosan/gelatin drug-loaded nanoparticles.
Example 7
Dissolving 1g of chitosan in 10mL of acetic acid aqueous solution with the mass fraction of 1%, adding 0.1g of etamsylate to prepare a mixed solution, filtering the mixed solution to remove insoluble substances after complete dissolution, taking 8mL of the filtered solution as a water phase, mixing liquid paraffin and petroleum ether according to a volume ratio of 1; the water phase and the oil phase are ultrasonically mixed according to the volume ratio of 1. Slowly dropwise adding 5mL of glutaraldehyde saturated toluene solution, keeping the temperature at 40 ℃, crosslinking under high-speed stirring (4000 r/min), centrifuging at 8000rpm, discarding supernatant, washing with petroleum ether, acetone and ethanol respectively, and freeze-drying to obtain chitosan drug-loaded nanoparticles with uniform particle size.
Example 8
Methacrylated chitosan was taken and dissolved in 10mL of distilled water containing 0.1% of etamsylate and 0.5% of lithium acylphosphinate. Dissolving in water bath at 40 deg.C to obtain mixed solution containing 15% of methacryloylated chitosan. 10mL of edible soybean oil and paraffin oil (volume ratio 1. The prepared solution was slowly dropped into the mixed oil in a stirred state while an ultraviolet curing lamp was placed under a beaker, stirred at high speed for 5 minutes while ultraviolet curing was performed, stirring was stopped, and the mixture was left to stand for five minutes. And then, respectively washing the nanoparticles by using precooled (5 ℃) dioxane, acetone and ethanol, fully removing residual oil on the surfaces of the nanoparticles, and drying the nanoparticles in a 60 ℃ oven overnight.
Example 9
The syringe was connected to the microfluidic device and the fluid was injected into both inlets using the propulsion device of the micro-injection pump. Mineral oil was used as the continuous phase and 5% (w/w) Span80 was used as the surfactant. A PBS solution containing 0.1% etamsylate and 1% methacrylated chitosan was mixed with a 0.5% photoinitiator solution in a volume ratio of 1. The syringe pump was started and the internal phase (flow rate 0.5 mL/min) and the continuous phase liquid (flow rate 1 mL/min) were injected into the apparatus. GelMA forms droplets due to the shear stress of the continuous phase liquid and is transferred from the outlet onto the cross-linked disk. The hydrogel droplets are photocrosslinked by ultraviolet irradiation to form solid gel nanoparticles. The hydrogel was washed with acetone and 75% alcohol to remove the surfactant and mineral oil, and lyophilized for storage.
Example 10
The liposome is prepared by a film dispersion method. 1g of phospholipid and 1g of cholesterol were dissolved in 10mL of a chloroform solution containing alpha-tocopherol (1 wt%) to obtain a lipid solution. Then adding 0.1g of etamsylate into 10mL of methanol to obtain an etamsylate solution, transferring the etamsylate solution into a constant-temperature water bath at 40 ℃ to mix the etamsylate solution with the lipoid solution, carrying out reduced pressure distillation by using a rotary evaporator to remove the organic solvent, forming a uniform lipoid film on the bottle wall, hydrating the lipoid film by using 10mL of hydration medium (40 ℃) dissolved with a freeze-drying protective agent to form a coarse suspension, carrying out vortex light for 2h, carrying out ultrasonic treatment for 10min, and storing in a refrigerator at 4 ℃.
Example 11
Adding 5g of soluble starch into a corresponding amount of 2mol/L NaOH and etamsylate solution (the mass percentage of the etamsylate is 1 percent), fully stirring, and activating for 30min at normal temperature. 10mL of soybean oil was added to the vessel, followed by 10mL of Span60, heated to 60 ℃ to completely dissolve the Span60, cooled to 40 ℃, and the prepared starch solution was added slowly with high speed stirring (4000 r/min). After liquid beads are completely and uniformly dispersed, adding 5mL of epoxy chloropropane, reacting for about 6h, performing centrifugal separation at 8000rpm, removing an oil phase, washing with ethyl acetate, repeatedly washing with ethanol and acetone, drying in a drying oven to obtain the drug-loaded starch nanoparticles, and performing freeze preservation.
Example 12
Taking a PBS (phosphate buffer solution) solution of the etamsylate (the mass percentage of the etamsylate is 0.1 wt%), adding a 1.5% sodium alginate solution (volume ratio is 3; uniformly mixing the corn oil, span-85 and tween-85 according to a volume ratio of 100. Slowly dripping the water phase into the oil phase by using a syringe under high-speed stirring (4000 r/min), and stirring for 5min to form a water/oil emulsion; then adding an ionic crosslinking agent solution (5% CaCl of the ionic crosslinking agent solution) 2 And 10% of ZnCl 2 The volume ratio of the ionic cross-linking agent solution to the phenolsulfonamine PBS solution is 1. Centrifuging at 8000rpm to separate nanoparticles, washing with sterilized water for injection for 3 times, and freeze-drying for storage.
Example 13
The syringe was connected to the microfluidic device and the fluid was injected into the three inlets using the propulsion device of the micro-injection pump. Using mineral oil as the continuous phase, a PBS solution containing etamsylate (0.1 wt% etamsylate) and sodium alginate (1.5 wt% sodium alginate) as the inner phase, 5% cacl 2 And 10% of ZnCl 2 The mixed aqueous solution of (2) serves as an external aqueous phase. The syringe pump was started and the external aqueous phase (flow rate 1 mL/min), internal phase (flow rate 0.5 mL/min) and continuous phase (flow rate 1 mL/min) were injected into the apparatus. Droplets are formed due to the shear stress of the continuous phase liquid. The hydrogel was washed with acetone and 75% alcohol to remove the surfactant and mineral oil, and lyophilized for storage.
Test example
Animal model experiments were performed on the etamsylate nano-formulation prepared in example 1 and a commercially available etamsylate general formulation. The rat spinal cord injury model is prepared by adopting a spinal cord contusion three-dimensional positioning device in a laboratory.
(1) Firstly, the end-point effect of the nano-preparation of etamsylate prepared in example 1 on treating spinal cord injury is observed, and behavioural tests are respectively carried out 1, 3, 7, 10, 14 and 21 days after the injury; namely, whether the nano-preparation of etamsylate prepared in example 1 can improve motor function recovery after spinal cord injury in rats. The test results are shown in fig. 1, and as a result, it was found that: compared with a blank control group and a common preparation hemostatic drug group, the nano preparation hemostatic drug can remarkably promote the BBB (Basso, beattie & Bresnahanolocomatoritingscale) score after the spinal cord injury of the rat.
(2) The test example further discusses the mechanism of the etamsylate nano preparation prepared in example 1 for promoting the recovery of the motor function, collects the injured spinal cord tissues, and adopts real-time quantitative PCR determination, because the etamsylate nano preparation prepared in example 1 is a foreign substance relative to the human body except the hemostatic efficacy of the drug itself, it is likely to be phagocytized by innate immune cells (microglia/macrophages) after entering the injured spinal cord, so the test example observes the influence of the etamsylate nano preparation on the local immune activation of the injury, and the result is shown in fig. 2, and the result shows that: compared with a control group and a common preparation hemostatic drug group, the nano preparation hemostatic drug can obviously reduce the transcription levels of proinflammatory immune factors TNF alpha, iNOS and IL-12 after spinal cord injury of rats;
the results shown in FIG. 3: compared with a control group and a common preparation hemostatic drug group, the nano preparation hemostatic drug can obviously increase the transcription levels of inflammatory-inhibiting immune factors Arginase1, ym1, IL-4 and IL-10 after spinal cord injury of rats.
(4) The test example examined the effect of the nano-preparation of etamsylate prepared in example 1 and the general preparation of etamsylate on the survival of neurons in the parainjury area after spinal cord injury and the number of nerve fibers, another index directly related to motor function recovery, in spinal rats. The test results are shown in fig. 4 and 5: compared with the common preparation hemostatic drug group, the nano preparation hemostatic drug can increase the number of residual nerve fibers around and in the damaged area after the spinal cord injury of the rat; compared with the common preparation hemostatic drug group, the nano preparation hemostatic drug can increase the survival of the neurons in the injury parazone after the spinal cord injury of the rat.
The results of the embodiment show that the nano-preparation of the etamsylate provided by the invention has the hemostatic effect of the drug per se to relieve secondary injury, and can improve the local immune microenvironment of injury after the nano-preparation reaches the local part of the injured tissue, so as to provide a good living environment for the survival of the peripheral neurons in the injury area, thereby promoting the survival of the peripheral neurons in the injury area, increasing the number of nerve fibers and further promoting the motor function recovery after spinal cord injury.
Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments belong to the protection scope of the present invention.
Claims (9)
1. Application of etamsylate in preparing medicine for treating spinal cord injury is provided.
2. The use of claim 1, wherein the treatment of spinal cord injury is hemostasis of spinal nerves.
3. The use according to claim 1 or 2, wherein the effective amount of etamsylate in the medicament is 0.1g/kg.
4. Use according to claim 1 or 2, wherein the etamsylate is a etamsylate nanoformulation.
5. The use according to claim 4, wherein the nano-preparation of etamsylate has a particle size of 60 to 100nm.
6. The use of claim 4, wherein the nano-formulation of etamsylate comprises etamsylate and a pharmaceutical carrier.
7. The use of claim 6, wherein the drug carrier comprises one or more of a polyester, chitosan, gelatin, photopolymerizable hydrogel, liposomes, starch, and sodium alginate.
8. Use according to claim 7, wherein the polyester comprises one or more of polycaprolactone, glycolide-L-lactide-caprolactone terpolymer, poly-DL-lactic acid-polyethylene glycol copolymer, poly-L-lactide and polylactic acid-glycolic acid copolymer.
9. The use of claim 7, wherein the photopolymerizable hydrogel comprises one or more of methacryloylated chitosan, methacryloylated gelatin, methacryloylated silk fibroin, methacryloylated hyaluronic acid, methacryloylated alginic acid, and methacryloylated dextran;
the liposome is prepared from phospholipid, cholesterol and alpha-tocopherol.
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| CN114099466A (en) * | 2021-11-24 | 2022-03-01 | 国家纳米科学中心 | Bionic cell membrane-inner core nano particle and preparation method and application thereof |
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| CN107441081A (en) * | 2017-08-23 | 2017-12-08 | 陕西医大血友病研究院 | A kind of complementary mixture for hemostasis for being used to treat hemophilia bleeding |
| CN114099466A (en) * | 2021-11-24 | 2022-03-01 | 国家纳米科学中心 | Bionic cell membrane-inner core nano particle and preparation method and application thereof |
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