CN106139174A - A kind of preparation method based on chitosan derivatives nanoscale ultrasound contrast agents - Google Patents
A kind of preparation method based on chitosan derivatives nanoscale ultrasound contrast agents Download PDFInfo
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- CN106139174A CN106139174A CN201610693711.0A CN201610693711A CN106139174A CN 106139174 A CN106139174 A CN 106139174A CN 201610693711 A CN201610693711 A CN 201610693711A CN 106139174 A CN106139174 A CN 106139174A
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- carboxymethyl chitosan
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 81
- 239000002961 echo contrast media Substances 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- -1 hexanoyl carboxymethyl Chemical group 0.000 claims abstract description 40
- 239000007788 liquid Substances 0.000 claims abstract description 30
- NBVXSUQYWXRMNV-UHFFFAOYSA-N fluoromethane Chemical compound FC NBVXSUQYWXRMNV-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000002872 contrast media Substances 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 15
- 239000000463 material Substances 0.000 claims abstract description 13
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 claims abstract description 8
- 238000004945 emulsification Methods 0.000 claims abstract description 6
- 239000007864 aqueous solution Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 229960004692 perflenapent Drugs 0.000 claims description 7
- NJCBUSHGCBERSK-UHFFFAOYSA-N perfluoropentane Chemical compound FC(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)F NJCBUSHGCBERSK-UHFFFAOYSA-N 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 4
- 238000001228 spectrum Methods 0.000 claims description 4
- 238000001157 Fourier transform infrared spectrum Methods 0.000 claims description 3
- 238000013019 agitation Methods 0.000 claims description 3
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 229960004624 perflexane Drugs 0.000 claims description 3
- ZJIJAJXFLBMLCK-UHFFFAOYSA-N perfluorohexane Chemical compound FC(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)F ZJIJAJXFLBMLCK-UHFFFAOYSA-N 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 2
- PKHMTIRCAFTBDS-UHFFFAOYSA-N hexanoyl hexanoate Chemical compound CCCCCC(=O)OC(=O)CCCCC PKHMTIRCAFTBDS-UHFFFAOYSA-N 0.000 claims description 2
- 238000000527 sonication Methods 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 4
- 231100000252 nontoxic Toxicity 0.000 abstract description 3
- 230000003000 nontoxic effect Effects 0.000 abstract description 3
- 150000003904 phospholipids Chemical class 0.000 abstract description 2
- 238000005917 acylation reaction Methods 0.000 abstract 1
- 239000002245 particle Substances 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000008232 de-aerated water Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 125000003104 hexanoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 239000000693 micelle Substances 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- ODIGIKRIUKFKHP-UHFFFAOYSA-N (n-propan-2-yloxycarbonylanilino) acetate Chemical group CC(C)OC(=O)N(OC(C)=O)C1=CC=CC=C1 ODIGIKRIUKFKHP-UHFFFAOYSA-N 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 241001597008 Nomeidae Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 208000034713 Spontaneous Rupture Diseases 0.000 description 1
- PRPAGESBURMWTI-UHFFFAOYSA-N [C].[F] Chemical compound [C].[F] PRPAGESBURMWTI-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 125000004202 aminomethyl group Chemical group [H]N([H])C([H])([H])* 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000006196 deacetylation Effects 0.000 description 1
- 238000003381 deacetylation reaction Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000002296 dynamic light scattering Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 210000003989 endothelium vascular Anatomy 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000002601 radiography Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012876 topography Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/225—Microparticles, microcapsules
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Acoustics & Sound (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Radiology & Medical Imaging (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Preparation (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
A kind of preparation method based on chitosan derivatives nanoscale ultrasound contrast agents; it is by acylation reaction, carboxymethyl chitosan to be modified; synthesize and there is amphipathic positive hexanoyl carboxymethyl chitosan; add liquid fluorocarbon on this basis, use ultrasonic emulsification method to prepare the nanoscale ultrasound contrast agents being made up of liquid fluorocarbon kernel and chitosan derivatives shell.Compared with prior art, the macromolecular material safety non-toxic used by the present invention, price is significantly lower than synthetic phospholipid, and contrast agent preparation technology is easy, and operating condition is gentle, has preferable potential application foreground.
Description
Technical field
The invention belongs to ultrasonic imaging diagnosis technical field, be specifically related to a kind of super based on chitosan derivatives nanoscale
The preparation method of sound contrast agent.
Background technology
Acoustic contrast agent is widely used to the diagnosis of clinical disease at present, substantially increases the accuracy rate of ultrasonic diagnosis,
It is " milestone " in ultrasound medicine development history.Relative to imaging modes such as conventional CT, MRI, ultrasonic contrast has noinvasive
The advantage such as radiationless, easy to operate, cheap, becomes the important means of diagnosing tumor on Present clinical.
And acoustic contrast agent is basis and the key of ultrasonic contrast safely and efficiently.At present conventional it is
Sonovue is the ultrasound microbubble contrast agent of representative, for lipid gassiness acoustic contrast agent, its less stable, is susceptible to spontaneous
Rupture.And its particle diameter has focused largely in 2～10 μ m, and capillary endothelium gap is about 380～780nm, the most often
With microcapsular ultrasound contrast agent be cannot penetration rate of blood endothelial tube gap and arrive tumor tissues, lack tumor tissue cell outer to blood vessel
Specificity, and for the easy missing inspection of tumor of weary blood supply, which greatly limits the ultrasonic contrast diagnosis energy to diseases such as tumors
Power.
Macromolecule polymer material has a good biocompatibility, the advantage such as biodegradable, and that applies that it prepares ultrasonic makes
Shadow agent, particle diameter distribution uniform, stability is high, and compressive property is preferable, it has also become the focus of acoustic contrast agent research at present.And grow
The perfluorocarbon compound of fluorocarbon chain is in a liquid state when low temperature, i.e. liquid fluorocarbon, when ambient pressure reduction or temperature are increased to its boiling
During point, it can occur liquid-gas phase transition, liquid become gas.Wherein, perflenapent boiling point at ambient pressure is 29 DEG C, and human body
Temperature is 37 DEG C, therefore it is a kind of fluorocarbon material being well suited to prepare liquid-gas phase transition type acoustic contrast agent.
Summary of the invention
Present invention aims to above-mentioned existing problems and deficiency, it is provided that a kind of parcel liquid fluorocarbon, preparation technology
Simplicity, safety non-toxic, cheap, the preparation side based on chitosan derivatives nanoscale ultrasound contrast agents of good biocompatibility
Method.
The technical scheme is that and be achieved in that:
Preparation method based on chitosan derivatives nanoscale ultrasound contrast agents of the present invention, this contrast agent is by liquid fluorocarbon
Core and chitosan derivatives shell are constituted, and the material of described chitosan derivatives shell is positive hexanoyl carboxymethyl chitosan, and it is special
Point is to comprise the following steps:
(1) positive hexanoyl carboxymethyl chitosan is synthesized；
(2) positive hexanoyl carboxymethyl chitosan sugar aqueous solution is configured；
(3) liquid fluorocarbon is added drop-wise in positive hexanoyl carboxymethyl chitosan sugar aqueous solution, carries out ultrasonic emulsification process, make liquid fluorocarbon
It is wrapped in positive hexanoyl carboxymethyl chitosan, i.e. obtains the nanoscale being made up of liquid fluorocarbon kernel and chitosan derivatives shell
Acoustic contrast agent.
Wherein, the concrete operation method of described step (1) is as follows:
(11) weigh 250mg carboxymethyl chitosan, after being dissolved in 20ml pure water and stirring 24h, add the stirring of 20ml methanol
Uniformly；
(12) under quick magnetic agitation, it is added dropwise over the n-caproic anhydride of 0.33ml, and maintains rotating speed at the stirring bar of 1000rpm
12 h are reacted under part；
(13) with ethanol water (25%, v/v) dialysis 24h, then dialysing 24h with pure water, lyophilization i.e. obtains modified
Chitosan derivatives positive hexanoyl carboxymethyl chitosan, and identified by FTIR spectrum and proton electromagnetic spectrum map analysis
This product.
The concrete operation method of described step (2) is as follows:
Take positive hexanoyl carboxymethyl chitosan 20mg～50mg and be scattered in 10ml pure water dissolving.
The concrete operation method of described step (3) is as follows:
(31) take positive hexanoyl carboxymethyl chitosan sugar aqueous solution, after ice bath processes 10min, add 100ul～200ul liquid fluorocarbon；
(32) under condition of ice bath, the positive hexanoyl carboxymethyl chitosan sugar aqueous solution adding liquid fluorocarbon is carried out at ultrasonic emulsification
Reason, i.e. obtains chitosan derivatives nanoscale ultrasound contrast agents, and is placed in by contrast agent in 4 DEG C of refrigerators standby；Described ultrasonic full
The following condition of foot: ultrasonic amplitude is 20%, and total sonication time is 2min, every ultrasonic startup 3s, intermittent time 1s.
The material of described liquid fluorocarbon kernel is perflenapent or perflexane.
The present invention compared with prior art, has the advantage that
Material used by the present invention is chitin modified rear prepared by natural polymers, and material safety is nontoxic, biological
The compatibility is good, price is less than synthetic phospholipid；And, the preparation technology of the present invention is easy, applied range, can be by polymerization
Thing is modified so that it is has targeting characteristic, is prepared as targeted ultrasound contrast agent；Meanwhile, this polymer can be used as medicine and
Genophore.
The present invention is further illustrated below in conjunction with the accompanying drawings.
Accompanying drawing explanation
Fig. 1 is the FTIR spectrum figure of positive hexanoyl carboxymethyl chitosan of the present invention.
Fig. 2 is the hydrogen nuclear magnetic resonance spectrogram of positive hexanoyl carboxymethyl chitosan of the present invention.
Fig. 3 is the cytotoxicity analysis figure of positive hexanoyl carboxymethyl chitosan of the present invention.
Fig. 4 is the transmission electron microscope picture of nanoscale ultrasound contrast agents of the present invention.
Fig. 5 is the grain size distribution of nanoscale ultrasound contrast agents of the present invention.
Fig. 6 is the potential diagram of nanoscale ultrasound contrast agents of the present invention.
Fig. 7 is the external supersonic image of nanoscale ultrasound contrast agents of the present invention.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further illustrated.
Embodiment 1: synthesis chitosan derivatives positive hexanoyl carboxymethyl chitosan
Weighing 250 mg carboxymethyl chitosans, the molecular weight of this carboxymethyl chitosan is 20 KD, is dissolved in 20ml pure water also
After stirring 24 h, add 20ml methanol and stir；Then under the conditions of quick magnetic agitation, it is added dropwise over 0.33 ml n-caproic acid
Acid anhydride, and stirring reaction 12h under 1000 rpm rotating speeds；Then dialyse 24 h with ethanol water (25%, v/v), then use pure water
Dialyse 24 h, and lyophilization i.e. obtains modified chitosan derivatives positive hexanoyl carboxymethyl chitosan, and by Fu
This product is identified in leaf infrared spectrum and proton electromagnetic spectrum map analysis.
The infrared spectrum of carboxymethyl chitosan and positive hexanoyl carboxymethyl chitosan is as it is shown in figure 1, due to carboxymethyl chitosan
Deacetylation is high, so corresponding to the bending vibration of N-H on primary amino radical, covering amide I band and the absworption peak of II band.Product exists
1500-1550cm-1Absworption peak occurs, and bands of a spectrum are remarkably reinforced, 2960cm-1And 2870cm-1Belong to the stretching vibration of methyl, its
Absworption peak strengthens, and illustrates that carbochain is already connected on amino.
The proton magnetic wave spectrogram of positive hexanoyl carboxymethyl chitosan is as in figure 2 it is shown, its chemical shift is 1.9-2.9ppm is second
Acylate group, chemical shift be 3.6-4.0ppm be sugar ring hydrogen position, 2.0 ppm are carboxymethyl chitosan-COCH3On
Hydrogen.Chemical shift 0.8ppm, 1.2 ppm, 1.5 ppm, 2.2ppm are for being connected on amino methyl and methylene on hexane.
Embodiment 2: the cytotoxicity experiment of positive hexanoyl carboxymethyl chitosan
The toxicity of A2780 cell is made by the positive hexanoyl carboxymethyl chitosan material being respectively provided with variable concentrations (1mg, 5mg, 10mg)
With.Align hexanoyl carboxymethyl chitosan material by the culture medium containing 10% serum to disperse, be configured to concentration 1 mg/ml respectively,
5 mg/ml, 10 mg/ml；A2780 cell recovered and passes on when it is to exponential phase of growth, carrying out cell counting, root
Cell density is adjusted according to needs；With 96 orifice plate cover plant cells, every porocyte suspension 100ul, 1*10 is inoculated in every hole respectively4Individual carefully
Born of the same parents, cultivate 24h in 37 DEG C of incubators；With PBS, plate inner cell is carried out, adds the positive hexanoyl carboxylic first of 3 kinds of variable concentrations
Base enclosure chitosan material, every kind of concentration arranges 6 multiple holes, and matched group adds the blank cultures without nanoparticle, cell and nanometer
Grain hatches 24 h；Configure 10% CCK-8 culture medium and draw the culture medium on former culture plate, add the CCK-8 containing 10% and arrive
Containing the culture medium of 10% serum, put back to incubator and continue to cultivate 1h.Full-automatic microplate reader measures absorbance A at 450 nm.
Calculate according to formula, cell survival rate=［ (experimental group OD value zeroing group OD value)/(matched group OD value-zeroing group OD
Value) ］ × 100%.
Fig. 3 result shows: along with material concentration increases, the cytotoxicity of positive hexanoyl carboxymethyl chitosan increases therewith, deposits
Motility rate reduces, but when increasing to 10mg/ml, its cell survival rate, still more than 90%, illustrates the biological safety of this material relatively
Good, without obvious vitro cytotoxicity effect.
Embodiment 3: preparing the nanoscale ultrasound contrast agents of positive hexanoyl carboxymethyl chitosan parcel liquid fluorocarbon, its step is such as
Under:
(1) synthesis chitosan derivatives positive hexanoyl carboxymethyl chitosan；
(2) configuring positive hexanoyl carboxymethyl chitosan sugar aqueous solution, its method is: take positive hexanoyl carboxymethyl chitosan 20 mg～50 mg
It is scattered in 10 ml pure water to dissolve and is formed as chitosan derivatives micellar aqueous solution, and chitosan derivatives micellar aqueous solution
Concentration is 2 mg/ml；
(3) take after positive hexanoyl carboxymethyl chitosan sugar aqueous solution ice bath processes 10 min, add 100 ul～200ul liquid fluorocarbons,
Described liquid fluorocarbon is perflenapent or perflexane, and in the present embodiment, liquid fluorocarbon is perflenapent, and positive hexanoyl carboxylic
Methyl chitosan is 1mg:10 μ l with the ratio of perflenapent；Then under condition of ice bath, to the chitosan adding liquid fluorocarbon
Derivant micellar aqueous solution carries out ultrasonic emulsification process, makes liquid fluorocarbon be wrapped in positive hexanoyl carboxymethyl chitosan, i.e. obtains
Chitosan derivatives nanoscale ultrasound contrast agents, is placed in 4 DEG C of refrigerators standby；
Described ultrasonic meet following condition: ultrasonic amplitude is 20%, and total ultrasonic time is 2min, ultrasonic startup 3s, the intermittent time
1s。
Embodiment 4: the sign of nanoscale ultrasound contrast agents
(1) optical microscope and transmission electron microscope is used to carry out surface topography and constitute observation
After nanoscale ultrasound contrast agents being diluted with normal saline, observe under inverted microscope, it be heated to 37 DEG C,
Can be observed to contrast agent droplet distribution good, smooth surface is transparent, and size is more uniform.Under transmission electron microscope, observation prepares
Acoustic contrast agent is rounded, and smooth surface is bright, as shown in Figure 4.
(2) particle diameter and current potential:
By the nanometer acoustic contrast agent of prepared parcel liquid fluorocarbon, after taking ultra-pure water dilution, with dynamic light scattering determination its
Particle diameter also uses laser particle analyzer to detect its Zeta potential, and result shows that its mean diameter is (205.9 ± 45.31) nm, such as Fig. 5
Shown in, Zeta potential value is (-31.9 ± 6.08) mV, as shown in Figure 6.
Embodiment 5: the external supersonic radiography experiment of the nanoscale ultrasound contrast agents of parcel perflenapent
Fill 37 DEG C of de aerated water in the sink, extract the acoustic contrast agent suspension of 4 ml to Sparta's suction pipe, placement with syringe
Preheating 5 min under de aerated water, ultrasonic probe is immersed in underwater and just to Sparta's suction pipe, surpasses by Philips IU-22 type
Audio clinic instrument (probe model L12-5) carries out ultra sonic imaging, is respectively compared at different carriers concentration (2mg-5mg), different PFP body
Long-pending ratio, under (1-2%), different time sections (0-12min) and same concentration, observes it different mechanical indexs (MI=0.4-0.6)
As effect.Echoenhance in visible Sparta suction pipe, becomes intensive choice refreshments shape echogram, as shown in Figure 7.
Additionally, the chitosan derivatives nanoscale ultrasound contrast agents prepared of the present invention finds: change chitosan and liquid
The ratio of fluorine carbon, ultrasonic power and time etc. all can affect the yield of contrast agent, particle diameter distribution and contrasting effects.
And, the present invention is to use nanoparticle self assembly mode to be prepared for having amphipathic chitosan derivatives micelle water
Solution (positive hexanoyl carboxymethyl chitosan sugar aqueous solution), has amphipathic chitosan by liquid fluorocarbon with this under cryogenic and spreads out
Biological micelle aqueous solution, uses ultrasonic cell disintegration instrument or ultrasonic washing unit to carry out emulsifying, is successfully prepared with amphiphilic
Property chitosan derivatives positive hexanoyl carboxymethyl chitosan be shell, parcel liquid fluorocarbon nanoscale ultrasound contrast agents.
The present invention is described by case study on implementation, but does not limit the invention, with reference to description of the invention,
Other changes of the disclosed embodiments, as the professional person for this area is readily apparent that, such change should belong to
Within the scope of the claims in the present invention limit.
Claims (5)
1. a preparation method based on chitosan derivatives nanoscale ultrasound contrast agents, this contrast agent by liquid fluorocarbon kernel and
Chitosan derivatives shell is constituted, and the material of described chitosan derivatives shell is positive hexanoyl carboxymethyl chitosan, and its feature exists
In comprising the following steps:
(1) positive hexanoyl carboxymethyl chitosan is synthesized；
(2) positive hexanoyl carboxymethyl chitosan sugar aqueous solution is configured；
(3) liquid fluorocarbon is added drop-wise in positive hexanoyl carboxymethyl chitosan sugar aqueous solution, carries out ultrasonic emulsification process, make liquid fluorocarbon
It is wrapped in positive hexanoyl carboxymethyl chitosan, i.e. obtains the nanoscale being made up of liquid fluorocarbon kernel and chitosan derivatives shell
Acoustic contrast agent.
Preparation method based on chitosan derivatives nanoscale ultrasound contrast agents the most according to claim 1, it is characterised in that
The concrete operation method of described step (1) is as follows:
(11) weigh 250mg carboxymethyl chitosan, after being dissolved in 20ml pure water and stirring 24h, add the stirring of 20ml methanol
Uniformly；
(12) under quick magnetic agitation, it is added dropwise over the n-caproic anhydride of 0.33ml, and maintains rotating speed at the stirring bar of 1000rpm
12 h are reacted under part；
(13) with ethanol water (25%, v/v) dialysis 24h, then dialysing 24h with pure water, lyophilization i.e. obtains modified
Chitosan derivatives positive hexanoyl carboxymethyl chitosan, and identified by FTIR spectrum and proton electromagnetic spectrum map analysis
This product.
Preparation method based on chitosan derivatives nanoscale ultrasound contrast agents the most according to claim 1, it is characterised in that
The concrete operation method of described step (2) is as follows:
Take positive hexanoyl carboxymethyl chitosan 20mg～50mg and be scattered in 10ml pure water dissolving.
Preparation method based on chitosan derivatives nanoscale ultrasound contrast agents the most according to claim 1, it is characterised in that
The concrete operation method of described step (3) is as follows:
(31) take positive hexanoyl carboxymethyl chitosan sugar aqueous solution, after ice bath processes 10min, add 100ul～200ul liquid fluorocarbon；
(32) under condition of ice bath, the positive hexanoyl carboxymethyl chitosan sugar aqueous solution adding liquid fluorocarbon is carried out at ultrasonic emulsification
Reason, i.e. obtains chitosan derivatives nanoscale ultrasound contrast agents, and is placed in by contrast agent in 4 DEG C of refrigerators standby；Described ultrasonic full
The following condition of foot: ultrasonic amplitude is 20%, and total sonication time is 2min, every ultrasonic startup 3s, intermittent time 1s.
Preparation method based on chitosan derivatives nanoscale ultrasound contrast agents the most according to claim 1, it is characterised in that:
The material of described liquid fluorocarbon kernel is perflenapent or perflexane.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108079323A (en) * | 2017-11-22 | 2018-05-29 | 广州医科大学附属第三医院 | A kind of chitosan derivatives support preparation method for Ultrasound mediated gene delivering |
| CN108743814A (en) * | 2018-08-14 | 2018-11-06 | 骆漫 | A kind of preparation method of anti-nasopharyngeal carcinoma Chinese medicine composition |
Citations (1)
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| CN108079323A (en) * | 2017-11-22 | 2018-05-29 | 广州医科大学附属第三医院 | A kind of chitosan derivatives support preparation method for Ultrasound mediated gene delivering |
| CN108743814A (en) * | 2018-08-14 | 2018-11-06 | 骆漫 | A kind of preparation method of anti-nasopharyngeal carcinoma Chinese medicine composition |
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