CN108114273A - A kind of perfluocarbon albumin nano granular and preparation method and application - Google Patents
A kind of perfluocarbon albumin nano granular and preparation method and application Download PDFInfo
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- CN108114273A CN108114273A CN201810108515.1A CN201810108515A CN108114273A CN 108114273 A CN108114273 A CN 108114273A CN 201810108515 A CN201810108515 A CN 201810108515A CN 108114273 A CN108114273 A CN 108114273A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/38—Albumins
- A61K38/385—Serum albumin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5015—Organic compounds, e.g. fats, sugars
Abstract
The invention discloses a kind of perfluocarbon albumin nano granular, including albumin and perfluorinate carbons compound, the albumin wraps up the perfluorinate carbons compound, and the mass ratio of the perfluorinate carbons compound and the albumin is 4 ~ 40:1.Perfluocarbon albumin nano granular of the present invention possesses good biological safety as a kind of new platelet suppressant drug.Platelet counts are greatly reduced in meeting primosome after administration, and blood platelet various functions are also suppressed significantly, and possess the therapeutic potential of preferable clinical practice and reality.
Description
Technical field
The invention belongs to biomedicine field, the preparation side of the albumin nano granular specifically inhibited based on platelet function
Method.
Background technology
Blood platelet is biologically active fritter kytoplasm, it derives from the crack releasing of ripe megacaryocyte kytoplasm, right
The realization of the processes such as organism physiology and pathologic hemostasis, thrombosis, inflammatory reaction, wound healing is significant.Blood is small
The process sticked together mutually between plate is platelet aggregation, it participates in physiological or pathologic hemostasis, and it belongs to blood coagulation
Process.Thrombocythemia can cause body abnormal, and piastrenemia is often white secondary to polycythemia, chronic granulocyte
Blood disease, acute bleeding or haemolysis, malignant tumour, myelofibrosis and Postsplenectomy etc..
Clinical investigation shows, the occurrence and development of malignant tumour be usually associated with platelet counts increase and activity it is bright
Aobvious rise, being proportionate property of the two.Correlative study also indicates that there is pathologic positive feedbacks between tumour cell and blood platelet
It adjusts.Hematoblastic activation increases the transfer invasion and attack with tumour cell, and growing multiplication complements each other, carried for the growth of tumour cell
For more suitable environment.By the further investigation to the two mechanism of action, the special micro-environment system energy of tumour cell is found
Stimulating platelet activation and proliferation, and activate blood platelet can discharge a variety of growth factors and cell factor inducing inflammatory reaction,
Promote to play facilitation in tumour growth, tumor invasion and transfer process.
Either perfluorocarbon compound is artificial off-white color liquid or lotion to perfluocarbon, is that a species is annular or straight
Chain organic compound has very high gas and liquid compatibility, can be transmitted oxygen and carbon dioxide, compound once by
It is used as artificial blood.Perfluorocarbon liquids have the critical function of human blood sample, can will be transported to human body from the oxygen of lung's inspiration
Each position, and the carbon dioxide discharged in vivo can be transported to lung and excreted, perfluocarbon conveying gas is single
Pure physical process completes the transmission of gas in flowing.In chemistry and biologically inert distribution type is not required, biology in perfluocarbon
Compatibility is good.Chinese patent CN201110238056.7 is by the systems such as perfluocarbon and recombinant human epidermal growth factor, antibacterial material
It is standby into gelling agent, for treating diabetes；Perfluorinate carbons material is total to by Chinese patent CN200610044479.4 with phosphatide
With being prepared into keeping solution, for normal oral, venoclysis, stomach perfusion, skin ulcer external application keeping；Chinese patent
Perfluocarbon is prepared into gelling agent by CN200980147692.X, and for local medical and cosmetic use, treatment wound is burnt
Wound improves skin tightness etc..
But up to the present, on containing the albumin preparation of perfluocarbon to the text in terms of platelet function inhibition
It offers patent and has no record and report.
There are two types of the common methods for preparing albumin nano granular at present, i.e. cross-linking method and high pressure homogenization.Cross-linking method needs
The larger crosslinking agent of toxicity is introduced, seriously affects drug safety.Requirement of the high pressure homogenization to equipment it is high and prepare white egg
It is weak that white nanoparticle contains ability.These deficiencies limit the application of albumin nano granular related preparations clinically.
The content of the invention
In view of the above-mentioned problems, to solve the defect of the prior art, one of the objects of the present invention is to provide a kind of perfluorinated
Carbon albumin nano granular has platelet function good inhibition, and biological safety is high.
In order to realize the technical purpose of the present invention, the technical scheme is that：
A kind of perfluocarbon albumin nano granular, including albumin and perfluorinate carbons compound, the albumin package
The mass ratio of the perfluorinate carbons compound, the perfluorinate carbons compound and the albumin is 4~40:1.
The perfluocarbon albumin nano granular, average grain diameter are 25nm~500nm, preferably 100nm~300nm.
The perfluorinate carbons compound is chain perfluoro-compound, annular perfluoro-compound, and heterocycle perfluoro-compound is halogenated complete
One or more in fluoride or nitrogenous perfluoro-compound, the wherein preferred tetrafluoromethane of chain perfluoro-compound, perfluoroethane, trifluoro
One or more in methane, octafluoropropane, R-4112 or perflexane；The preferred perfluorodecalin of annular perfluoro-compound, eight
One or more in fluorine cyclobutane or perfluor 1,4- dimethyleyelohexane phenylpiperidines；The preferably perfluorinated 1,3- bis- of heterocycle perfluoro-compound
One or more in methyl adamantane, fluorination bicyclononane；The halogenated preferred perfluorooctyl bromide of perfluoro-compound；Nitrogenous perfluoro-compound
It is preferred that the one or more in perfluorotributylamine or perfluamine.
The albumin is seralbumin, preferably human serum albumins, bovine serum albumin(BSA), albumin rabbit serum,
One or more in porcine serum albumin, sheep blood serum albumin, donkey serum albumin or horse serum albumin.
The present invention also provides the preparation methods of above-mentioned perfluocarbon albumin nano granular, comprise the following steps：
By perfluorinate carbons compound and albumin solution according to perfluorinate carbons compound and albumin mass ratio 4~
40:1 is premixed, and is added in and helped emulsion, and under external force, perfluorinate carbons compound is contained into albumin, is formed white
Protein nano grain.
The concentration of the albumin solution is 1~20g/100mL.
The solvent of the albumin solution be water, saline citrate, phosphate aqueous solution, acetate aqueous solution,
Tartrate aqueous solution, lactic acid saline solution, sodium-chloride water solution, glucose solution, aqueous sucrose solution, Sodium Caprylate are water-soluble
One or more in liquid, acetyltryptophan aqueous solution, glycerine, mannitol, sorbierite, amino acid are preferably water, sodium chloride water
One or more in solution or glucose solution.
Further, the albumin solution is formed in 0 DEG C~65 DEG C latitude of formulation, preferably prepares temperature
Scope is 20 DEG C~35 DEG C.
Described helps emulsion as the one or more in ethyl alcohol, ethylene glycol, propylene glycol, glycerine or polyglycerol ester.
The external force act as ultrasound, at a high speed high pressure, 0 DEG C~65 DEG C of concussion, radiation or temperature.
Preferably, control system pH is 3~9, preferably 5~8.
The present invention also provides the perfluocarbon albumin nano granulars to prepare the application in inhibiting platelet function drug.
Perfluocarbon albumin nano granular of the present invention is in use, aqueous solution or freeze-dried powder combination can be made into
Object exists preferably in the form of freeze-dried powder composition.
Perfluocarbon albumin nano granular freeze-dried powder composition when in use, can with it is pharmaceutically acceptable, can phase
Any medium hold and injectable dissolves immediately and/or dilution.Preferred freeze-dried powder composition can use water for injection,
5% glucose injection dissolves and/or dilution.
Perfluocarbon albumin nano granular described in technical solution of the present invention, it is secondary available for inhibiting myeloproliferative disorders
Property piastrenemia, thrombus, tumour or other blood platelets increase position platelet function.
The inhibition platelet function inhibits hematoblastic activation degranulation, adherency, aggregation to reduce platelet counts
With the release for inhibiting the related activation factor.
Perfluocarbon albumin nano granular provided by the invention and preparation method and application, compared with prior art, tool
It has the advantage that：
The use of prior art toxicity crosslinking agent is avoided in preparation process of the present invention, improves the bio-safety of preparation
Property.
The preparation method of the present invention is low for equipment requirements, and woth no need to large-scale high-end devices, preparation process is simple and easy to do, has
The potentiality of large-scale production.
The present invention is innovatively found that perfluocarbon albumin nano granular possesses hematoblastic function and preferably inhibits to make
With the research and development for new antiplatelet disease medicament provide new approaches.
This is for treating with having great clinical research meaning for thrombocythemia relevant disease, especially for shortage
The clinical malignant tumour of effective medicine opens a new therapy approach.
Description of the drawings
Fig. 1 is the TEM phenogram pictures of the albumin nano granular prepared by embodiment 1.
Fig. 2 is the dynamic light scattering grain size comparison diagram for the perfluocarbon albumin nano granular that embodiment 13 is tested.
Fig. 3 is the plasma stability comparison diagram for the perfluocarbon albumin nano granular that embodiment 14 is tested.
Fig. 4 is to blood platelet in embodiment 15 with the albumin nano granular prepared by clot retraction experiment investigation embodiment 1
The influence comparison diagram of quantity.
Fig. 5 is to blood platelet in embodiment 16 with the albumin nano granular prepared by clot retraction experiment investigation embodiment 1
The influence comparison diagram of function.
Fig. 6 is to blood platelet in embodiment 17 with the albumin nano granular prepared by docking clotting assay investigation embodiment 1
The influence comparison diagram of function.
Fig. 7 is to investigate perfluocarbon albumin nano granular to platelet adhesion reaction work(using Laser Scanning Confocal Microscope in embodiment 18
The influence comparison diagram of energy.
Fig. 8 is the cytotoxicity controls figure of perfluocarbon albumin nano granular in embodiment 19.
Specific embodiment
The present invention is further illustrated with reference to example, implementation below only describes this hair by way of example
It is bright.But it does not mean that and the present invention is limited.Those skilled in the art are spiritual without prejudice to the invention
On the premise of can also make a variety of equivalent modifications or replacement, these equivalent modifications or replacement are all contained in the application right will
It asks in limited range.It is to be understood that this invention is intended to cover the accommodation included in the dependent claims and repair
Change.
Embodiment 1
The preparation of perfluorotributylamine (PFTBA)-albumin nano granular：
Human serum albumins stoste (10g/50mL) 0.5mL is measured into 7mL cillin bottles, deionized water is added in and is diluted to
2mL, in 20 DEG C of mixings.1.5mL perfluorotributylamines (PFTBA) are added, add in 1.0mL ethyl alcohol (analysis is pure), mixing afterwards.Make
Go deep into inside cillin bottle, using the power of 500W when ultrasonic, setting and stopping per after 2s ultrasounds to close to liquid bottom with ultrasonic probe
Only 3s, ultrasound 3 times, 2 minutes every time, ultrasound end every time needed preparation standing 1-2 minutes in 20 DEG C of water-baths.Sample is turned
It moves in 4mL centrifuge tubes, 3000rpm centrifugation 3min take supernatant, the perfluorotributylamine albumin nano granular as prepared.
Three fourth of perfluor is observed by transmission electron microscope (transmission electron microscope, TEM)
The form of amine-albumin nano granular.First, by 160 times of the preparation diluent of preparation, the copper mesh for having been coated with agraphitic carbon is taken to receive
It infiltrates 2-3 times, is placed in the culture dish for being placed with filter paper repeatedly in grain of rice lotion, spontaneously dry overnight.After for 24 hours, sample is put
Enter in transmission electron microscope sample slot, observed.The results show that the nanoparticle has apparent albumin layer structure, and put down
Equal grain size is 170nm, sees Fig. 1.
Embodiment 2
The preparation of perfluorooctyl bromide (PFOB)-albumin nano granular：
It is 5g/100mL to measure human serum albumins stoste and add in water to be diluted to albumin concentration, in 25 DEG C of mixings.It takes
The solution 2mL after dilution is stated, adds in 3g perfluorooctyl bromide (PFOB), adds in 1.0mL ethyl alcohol (analysis is pure), mixing afterwards.It uses
Ultrasonic probe gos deep into inside bottle being ultrasonically treated to close to liquid bottom.Sample is transferred in centrifuge tube, 3000rpm centrifugations
3min takes supernatant, the perfluorooctyl bromide albumin nano granular as prepared, average grain diameter 220nm.
Embodiment 3
The preparation of perfluamine (PFTPA)+perfluorodecalin (FDC)-albumin nano granular：
It is 5g/100mL to measure human serum albumins stoste and add in water to be diluted to albumin concentration, in 30 DEG C of mixings.It takes
The solution 2mL after dilution is stated, adds in 0.33g perfluamines and 0.79g perfluorodecalins, adds in (the analysis of 1.0mL ethyl alcohol afterwards
It is pure), mixing.Go deep into inside bottle being ultrasonically treated to close to liquid bottom using ultrasonic probe.Sample is transferred to centrifuge tube
In, 3000rpm centrifugation 3min take supernatant, the perfluamine perfluorodecalin albumin nano granular as prepared, and average grain diameter is
150nm。
Embodiment 4
The preparation of perfluor 1,4- dimethyleyelohexanes phenylpiperidines (FMCP)+perfluorodecalin (FDC)-albumin nano granular：
It is 5g/100mL to measure human serum albumins stoste and add in water to be diluted to albumin concentration, in 35 DEG C of mixings.It takes
The solution 2mL after dilution is stated, adds in 200 μ L perfluors 1,4- dimethyleyelohexanes phenylpiperidines and 400 μ L perfluorodecalins add in afterwards
1.0mL ethyl alcohol (analysis is pure), mixing.Go deep into inside bottle being ultrasonically treated to close to liquid bottom using ultrasonic probe.By sample
Product are transferred in centrifuge tube, and 3000rpm centrifugation 3min take supernatant, the perfluor Isosorbide-5-Nitrae-dimethyleyelohexane phenylpiperidines as prepared are complete
Fluoronaphthalene alkane albumin nano granular, average grain diameter 230nm.
Embodiment 5
The preparation of tetrafluoromethane albumin nano granular：
It is 1g/100mL to measure human serum albumins stoste and add in aqueous citric acid solution to be diluted to albumin concentration, in 0 DEG C
Mixing.The solution 10mL after above-mentioned dilution is taken, adds in 1.0mL ethylene glycol (analysis is pure), mixing, it is 3 to control pH, by above-mentioned solution
It is placed in closed container, is filled with 4g tetrafluoromethanes, control mixed solution is uniformly mixed under high pressure.Afterwards by sample be transferred to from
In heart pipe, 3000rpm centrifugation 3min take supernatant, the tetrafluoromethane albumin nano granular as prepared, average grain diameter 300nm.
Embodiment 6
The preparation of octafluorocyclobutane albumin nano granular：
It is 10g/100mL to measure bovine serum albumin(BSA) stoste and add in phosphate aqueous solution to be diluted to albumin concentration, in 10
DEG C mixing.The solution 1mL after above-mentioned dilution is taken, adds in 3g octafluorocyclobutanes, 1.0mL propylene glycol (analysis is pure) is added in afterwards, mixes
Even, it is 5 to control pH.Sample is transferred in centrifuge tube after shaking at a high speed, 3000rpm centrifugation 3min take supernatant, as prepare
Octafluorocyclobutane albumin nano granular, average grain diameter 500nm.
Embodiment 7
The preparation of perfluoroethane+perfluorinated 1,3- dimethyladamantane albumin nano granulars：
It is 7g/100mL to measure albumin rabbit serum stoste and add in sodium-chloride water solution to be diluted to albumin concentration, in 15 DEG C
Mixing.The solution 2mL after above-mentioned dilution is taken, adds in perfluorinated 1, the 3- dimethyladamantanes of 3g, 1.0mL glycerine is added in afterwards and (divides
Analyse pure), mixing, it is 7 to control pH, and above-mentioned solution is placed in closed container, is filled with 1.9g perfluoroethanes.Afterwards to entirely reacting
After system carries out high speed concussion, sample is transferred in centrifuge tube, 3000rpm centrifugation 3min take supernatant, the hexafluoro as prepared
Perfluorinated 1, the 3- dimethyladamantanes albumin nano granular of ethane, average grain diameter 430nm.
Embodiment 8
The preparation of fluoroform albumin nano granular：
It is 3g/100mL to measure porcine serum albumin stoste and add in glucose solution to be diluted to albumin concentration, in 50 DEG C
Mixing.The solution 5mL after above-mentioned dilution is taken, adds in 1.0mL polyglycerol esters (analysis is pure) afterwards, mixing, it is 8 to control pH, will be upper
It states solution to be placed in closed container, is filled with 2.55g fluoroforms.After entire reaction system is shaken mixing at 65 DEG C afterwards,
Sample is transferred in centrifuge tube, 3000rpm centrifugation 3min take supernatant, and the fluoroform albumin nano granular as prepared is put down
Equal grain size is 330nm.
Embodiment 9
The preparation of octafluoropropane albumin nano granular：
It is 1g/100mL to measure sheep blood serum albumin stoste and add in amino acid solution to be diluted to albumin concentration, mixed in 65 DEG C
It is even.The solution 10mL after above-mentioned dilution is taken, adds in 1.0mL ethyl alcohol (analysis is pure), mixing, it is 5 to control pH, and above-mentioned solution is placed in
In closed container, 1.5g octafluoropropanes are filled with.Sample is transferred in centrifuge tube after 30 DEG C of stirrings, 3000rpm centrifugation 3min,
Supernatant is taken, the octafluoropropane albumin nano granular as prepared, average grain diameter 120nm.
Embodiment 10
The preparation of R-4112 albumin nano granular：
It is 12g/100mL to measure donkey serum albumin stoste and add in glycerine to be diluted to albumin concentration, in 25 DEG C of mixings.It takes
Solution 5mL after above-mentioned dilution adds in 6g R-4112s, adds in 1.0mL ethyl alcohol (analysis is pure) afterwards, mixing, controls the pH to be
9.Sample is transferred in centrifuge tube after 0 DEG C of stirring, 3000rpm centrifugation 3min take supernatant, the R-4112 as prepared
Albumin nano granular, average grain diameter 100nm.
Embodiment 11
The preparation of perflexane albumin nano granular：
It is 20g/100mL to measure horse serum albumin stoste and add in lactic acid saline solution to be diluted to albumin concentration, in 5 DEG C
Mixing.The solution 1mL after above-mentioned dilution is taken, adds in 1g perflexanes, adds in 1.0mL ethyl alcohol (analysis is pure), mixing, control afterwards
PH is 7.Sample is transferred in centrifuge tube after 10 DEG C of stirrings, 3000rpm centrifugation 3min take supernatant, the perfluor as prepared
Hexane albumin nano granular, average grain diameter 60nm.
Embodiment 12
It is fluorinated the preparation of bicyclononane albumin nano granular：
It is 5g/100mL to measure porcine serum albumin stoste and add in mannitol to be diluted to albumin concentration, in 40 DEG C of mixings.
The solution 5mL after above-mentioned dilution is taken, adds in 1g fluorination bicyclononane, adds in 1.0mL ethyl alcohol (analysis is pure), mixing, control afterwards
PH is 8.Sample is transferred in centrifuge tube after 25 DEG C of stirrings, 3000rpm centrifugation 3min take supernatant, the fluorination as prepared
Bicyclononane albumin nano granular, average grain diameter 25nm.
Embodiment 13
The diameter characterization of perfluocarbon-albumin nano granular
(concentration is the perfluocarbon-albumin nano granular prepared with dynamic light scattering (DLS) detection embodiment 1,2,3 and 4
Grain size 3mg/mL) takes 2mL dilution Nanobrook 90Plus Zata nanoparticle analyzers.The result shows that measure grain
Between footpath its particle size of size the results show is about 150-230nm, Fig. 2 is seen.
Embodiment 14
The plasma stable Journal of Sex Research of perfluocarbon-albumin nano granular
Particle size might have slight variations after nanoparticle places a period of time in different environments.Therefore embodiment
1st, in Swine plasma, (commercially available pig blood adds in anticoagulant heparin, 5000rpm to 2, the 3 and 4 perfluocarbon-albumin nano granulars prepared in advance
Centrifuge 10min, centrifugation can obtain twice) in 40 times of dilution, be placed on 4 DEG C, respectively in 0h, 1h, 2h, 3h, 5h, 8h, use for 24 hours
Nanobrook 90Plus Zata nanoparticle analysis-e/or determining grain sizes.Nanoparticle stoste is also disposed in 4 DEG C, at more than time point
Stoste distilled water is taken to dilute 40 times of measure grain sizes, sees Fig. 3.
Embodiment 15
Perfluocarbon-albumin nano granular influences to test on platelet counts
The Balb/c mouse of health is taken to be divided into 2 groups, every group 8, i.e., experimental group for the preparation of embodiment 1 perfluorotributylamine-
Albumin nano granular (300mg/mL) and control group (physiological saline).Tail vein injection method is taken, 0.1mL is administered every time.
After administration after 10h, each group Mouse whole blood is taken to do blood routine, detect the variation of platelet counts in Mice Body, see Fig. 4.As a result table
Bright, the hematoblastic quantity conspicuousness of perfluorotributylamine-albumin nano granular experimental mice is reduced, and sees Fig. 4.
Embodiment 16
The docking clotting assay of perfluocarbon-albumin nano granular
The Balb/c mouse of health is taken to be divided into 2 groups, every group 8, i.e., experimental group for the preparation of embodiment 1 perfluorotributylamine-
Albumin nano granular (300mg/mL) and control group (physiological saline).Tail vein injection method is taken, 0.1mL is administered every time.
After administration after 10h, it will be cut respectively with scissors at Mouse Tail-tip 0.5cm, blood starts timing after voluntarily flowing out, and is used every 5min
Filter paper sucks drop of blood once, until blood stops naturally, calculates the bleeding time in bleeding time, experiment with computing group and control group,
And it is compared.The result shows that the clotting time of experimental group is considerably longer than control group, it is seen that perfluorotributylamine-albumin nano
Grain can inhibit platelet function, postpone the clotting time, see Fig. 5.
Embodiment 17
The clot retraction experiment of perfluocarbon-albumin nano granular
Rabbit heart takes blood, and the rabbit blood of taking-up is put into the 50mL silication centrifuge tubes for being previously added 4% sodium citrate anti-freezing.
Centrifuge tube is put into centrifuge, 1000rpm centrifuges 5min, gives up the red blood cell layer of lower floor, and Aspirate supernatant is placed again into centrifugation
Machine 1000rpm centrifuges 5min, and gained supernatant is platelet rich plasma (PRP).Take the embodiment 1/ of the PRP and 20 μ L of 80 μ L
2/3/4 perfluocarbon-the albumin preparation prepared (HEPES is control) mixing, is put in 37 DEG C of cell incubators and is incubated altogether
30min.After incubation, the PRP blended liquids of 95 μ L are added in 1.5mL silication centrifuge tubes, the red blood cell of 5 μ L adds in 890 μ L
HEPES dilutions, be eventually adding the 10 μ L of fibrin ferment of 100U/mL, and be rapidly inserted into capillary, after waiting for a period of time, treat blood
Clotting collection is shunk, and finally measures clot quality and Serology Quality, characterizes biologically active pdgf.The experimental results showed that perfluocarbon-white
To some extent than control group (NaCl groups) slowly, the quality of retraction clot is less than control to the clot retraction speed of protein groups
Group, Serology Quality are more than control group.Illustrate that perfluocarbon-albumin preparation can inhibit platelet function to a certain extent,
Delay clot retraction process, see Fig. 6.
Embodiment 18
The platelet attachment experiment of perfluocarbon-albumin nano granular
The Balb/c mouse orbits of health is taken to take blood, add in (the volume ratio 1 containing 4% sodium citrate:9) 1.5mL of anti-freezing from
Heart pipe is put into centrifuge 1000rpm centrifugation 6min, abandons lower sediment, gained supernatant 1000rpm centrifuges 6min to go again
Except other haemocytes that may be present, it is platelet rich plasma (PRP) to take supernatant.Take the PRP and 20 μ LHEPES or reality of 80 μ L
Perfluorotributylamine-albumin nano granular preparation mixing of the preparation of example 1 is applied, 30min is incubated at 37 DEG C.Blended liquid is dropped in into glass slide
Upper (having drawn a circle with vaseline in advance, prevent that liquid from scattering exsiccations), 37 DEG C are incubated 30min, glass slide PBS flushings 3 times,
100 μ LDIR (20 μ L of saturated solution are diluted to 1mL) are eventually adding, 10min is incubated at room temperature, sucks dyeing liquor, add a drop PBS, altogether
It is observed under focusing microscope.The result shows that the platelet adhesion reaction quantity of perfluorotributylamine-albumin group is considerably less than control group,
Stick it can be seen that perfluorotributylamine-albumin nano granular can inhibit platelet activation, see Fig. 7.
Embodiment 19
The cytotoxicity of perfluocarbon-albumin nano granular
Good biological safety is the premise of pharmaceutical preparation application, and the present embodiment uses Human umbilical vein endothelial cells
(HUVEC) model, under the conditions of investigating various concentration, by the cytotoxicity of albumin nano granular prepared by embodiment 1/2/3/4.It will
Human umbilical vein endothelial cells are inoculated in 96 orifice plates with 5000/ cell density per hole, are incubated overnight adherent.PFTBA@HSA are used
Culture medium is diluted by concentration gradient, and 100 μ L various concentration drugs are added in per hole, and each concentration sets 3 secondary orifices, and dosing is placed in training
Foster case takes out cell plates after being incubated 6h altogether, siphons away the drug in hole, with PBS cleaning twice.The CCK-8 of 100 μ L is added in per hole,
It is incubated under the conditions of 37 DEG C.After color change, absorbance under 450nm is detected with microplate reader, using untreated cell as reference, is calculated
Cell survival rate.The experimental results showed that a series of perfluocarbon albumin nano granulars that the present invention designs are in various concentration drug
Under the action of, cell survival rate is close consistent with control group, illustrates that the perfluocarbon albumin nano granular has good life
Object security, is shown in Fig. 8.
Claims (10)
1. a kind of perfluocarbon albumin nano granular, which is characterized in that described white including albumin and perfluorinate carbons compound
Perfluorinate carbons compound described in protein encapsulation, the mass ratio of the perfluorinate carbons compound and the albumin for 4 ~
40:1。
2. nanoparticle according to claim 1, which is characterized in that the average grain diameter of the nanoparticle for 25nm ~
500nm, preferably 100nm ~ 300nm.
3. nanoparticle according to claim 1, which is characterized in that the perfluorinate carbons compound is perfluorinated for chain
One or more in object, annular perfluoro-compound, heterocycle perfluoro-compound, halogenated perfluoro-compound or nitrogenous perfluoro-compound, wherein chain
One in the preferred tetrafluoromethane of shape perfluoro-compound, perfluoroethane, fluoroform, octafluoropropane, R-4112 or perflexane
Kind is a variety of；One in the preferred perfluorodecalin of annular perfluoro-compound, octafluorocyclobutane or perfluor 1,4- dimethyleyelohexane phenylpiperidines
Kind is a variety of；One or more in the preferably perfluorinated 1,3- dimethyladamantanes of heterocycle perfluoro-compound, fluorination bicyclononane；Halogen
For the preferred perfluorooctyl bromide of perfluoro-compound；The preferred perfluorotributylamine of nitrogenous perfluoro-compound or one kind or more in perfluamine
Kind.
4. nanoparticle according to claim 1, which is characterized in that the albumin is seralbumin, preferably people's blood
Pure albumen, bovine serum albumin(BSA), albumin rabbit serum, porcine serum albumin, sheep blood serum albumin, donkey serum albumin or horse
One or more in seralbumin.
5. a kind of preparation method of any perfluocarbon albumin nano granulars of claim 1-4, which is characterized in that including with
Lower step：
By perfluorinate carbons compound and albumin solution according to perfluorinate carbons compound and albumin mass ratio 4 ~ 40:1 into
Row premix, and add in and help emulsion, under external force, perfluorinate carbons compound is contained into albumin, forms albumin nanometer
The grain of rice.
6. according to the method described in claim 5, it is characterized in that, the solvent of the albumin solution is water, citrate
Aqueous solution, phosphate aqueous solution, acetate aqueous solution, tartrate aqueous solution, lactic acid saline solution, sodium-chloride water solution, Portugal
Grape sugar aqueous solution, aqueous sucrose solution, sad sodium water solution, acetyltryptophan aqueous solution, glycerine, mannitol, sorbierite, amino acid
In one or more, be preferably water, sodium-chloride water solution or glucose solution in one or more.
7. according to the method described in claim 5, it is characterized in that, described help emulsion as ethyl alcohol, ethylene glycol, propylene glycol, sweet
One or more of oil or polyglycerol ester.
8. according to the method described in claim 5, it is characterized in that, the external force act as ultrasound, high pressure, at a high speed concussion,
0 DEG C ~ 65 DEG C of radiation or temperature.
9. according to the method described in claim 5, it is characterized in that, control system pH is 3 ~ 9, preferably 5 ~ 8.
10. a kind of any perfluocarbon albumin nano granulars of claim 1-4 are preparing the medicine of inhibition platelet function
Application in object.
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| CN111214459A (en) * | 2020-03-13 | 2020-06-02 | 南京大学 | Perfluorocarbon albumin nanoparticles and application thereof in preparation of tumor treatment drugs |
| CN114617839A (en) * | 2022-03-31 | 2022-06-14 | 温州医科大学 | Gel liniment for treating frozen cold injury and preparation method thereof |
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| CN111214459A (en) * | 2020-03-13 | 2020-06-02 | 南京大学 | Perfluorocarbon albumin nanoparticles and application thereof in preparation of tumor treatment drugs |
| CN111214459B (en) * | 2020-03-13 | 2022-09-23 | 南京大学 | Perfluorocarbon albumin nanoparticles and application thereof in preparation of tumor treatment drugs |
| CN114617839A (en) * | 2022-03-31 | 2022-06-14 | 温州医科大学 | Gel liniment for treating frozen cold injury and preparation method thereof |
| CN114617839B (en) * | 2022-03-31 | 2023-07-07 | 温州医科大学 | Gel liniment for treating freezing cold injury and preparation method thereof |
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