CN106139174B - 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
- Publication number
- CN106139174B CN106139174B CN201610693711.0A CN201610693711A CN106139174B CN 106139174 B CN106139174 B CN 106139174B CN 201610693711 A CN201610693711 A CN 201610693711A CN 106139174 B CN106139174 B CN 106139174B
- Authority
- CN
- China
- Prior art keywords
- carboxymethyl chitosan
- chitosan
- positive hexanoyl
- liquid fluorocarbon
- chitosan derivatives
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229920001661 Chitosan Polymers 0.000 title claims abstract description 76
- 239000002961 echo contrast media Substances 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- -1 hexanoyl carboxymethyl Chemical group 0.000 claims abstract description 40
- 239000007788 liquid Substances 0.000 claims abstract description 31
- NBVXSUQYWXRMNV-UHFFFAOYSA-N fluoromethane Chemical compound FC NBVXSUQYWXRMNV-UHFFFAOYSA-N 0.000 claims abstract description 25
- 239000002872 contrast media Substances 0.000 claims abstract description 19
- 239000000463 material Substances 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 11
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 claims abstract description 9
- 238000004945 emulsification Methods 0.000 claims abstract description 5
- 239000007864 aqueous solution Substances 0.000 claims description 14
- 238000002604 ultrasonography Methods 0.000 claims description 10
- 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 8
- 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
- 238000000502 dialysis Methods 0.000 claims description 6
- 238000001157 Fourier transform infrared spectrum Methods 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 4
- 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
- PKHMTIRCAFTBDS-UHFFFAOYSA-N hexanoyl hexanoate Chemical compound CCCCCC(=O)OC(=O)CCCCC PKHMTIRCAFTBDS-UHFFFAOYSA-N 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
- 238000010183 spectrum analysis Methods 0.000 claims description 3
- 229920001282 polysaccharide Polymers 0.000 claims description 2
- 238000000527 sonication Methods 0.000 claims description 2
- 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
- 206010028980 Neoplasm Diseases 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- PRPAGESBURMWTI-UHFFFAOYSA-N [C].[F] Chemical compound [C].[F] PRPAGESBURMWTI-UHFFFAOYSA-N 0.000 description 3
- 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
- 230000000694 effects 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
- 230000001965 increasing effect Effects 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 239000002105 nanoparticle Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 229910052799 carbon Inorganic materials 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
- 238000002474 experimental method Methods 0.000 description 2
- 229910052731 fluorine Inorganic materials 0.000 description 2
- 239000011737 fluorine Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229920000642 polymer Polymers 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
- 239000000243 solution Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 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
- 108010087230 Sincalide Proteins 0.000 description 1
- 208000034713 Spontaneous Rupture Diseases 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
- 238000004458 analytical method Methods 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 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
- 125000000837 carbohydrate group Chemical group 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 239000008232 de-aerated water Substances 0.000 description 1
- 230000006196 deacetylation Effects 0.000 description 1
- 238000003381 deacetylation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000002296 dynamic light scattering Methods 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 210000003989 endothelium vascular Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 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 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 238000002329 infrared spectrum Methods 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
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 238000000399 optical microscopy Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 238000002601 radiography Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 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 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012876 topography Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 239000003643 water by type Substances 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 to be modified by acylation reaction to carboxymethyl chitosan; having synthesized has amphiphilic positive hexanoyl carboxymethyl chitosan; liquid fluorocarbon is added on this basis, the nanoscale ultrasound contrast agents being made of liquid fluorocarbon kernel and chitosan derivatives shell are made using ultrasonic emulsification method.Compared with prior art, the high molecular material used in the present invention is safe and non-toxic, and price is significantly lower than synthetic phospholipid, and contrast agent preparation process is easy, and operating condition is mild, has preferable potential application foreground.
Description
Technical field
The invention belongs to ultrasonic imaging diagnosis technical fields, are specifically related to a kind of super based on chitosan derivatives nanoscale
The preparation method of sound contrast agent.
Background technique
Acoustic contrast agent is widely used to the diagnosis of clinical disease at present, substantially increases the accuracy rate of ultrasound diagnosis,
It is one " milestone " in ultrasound medicine development history.Relative to imaging modes such as conventional CT, MRI, ultrasonic contrast possesses noninvasive
The advantages such as radiationless, easy to operate, cheap, become the important means of diagnosing tumor on Present clinical.
And safe and efficient acoustic contrast agent is basis and the key of ultrasonic contrast.At present clinically the most commonly used is
Sonovue is the ultrasound microbubble contrast agent of representative, is lipid gassiness acoustic contrast agent, stability is poor, is easy to happen spontaneous
Rupture.And its partial size has focused largely in 2~10 μ ms, and capillary endothelium gap is about 380~780nm, therefore often
It is that can not penetrate vascular endothelial gap and reach tumor tissues with microcapsular ultrasound contrast agent, lacks to tumor tissue cell outside blood vessel
Specificity, and for the tumour of weary blood supply be easy missing inspection, which greatly limits ultrasonic contrasts to the diagnosis energy of the diseases such as tumour
Power.
Macromolecule polymer material has many advantages, such as good biocompatibility, biodegradable, makes using its ultrasound prepared
Shadow agent, particle diameter distribution is more uniform, and stability is high, and compressive property is preferable, it has also become the hot spot of acoustic contrast agent research at present.And it grows
The perfluorocarbon compound of fluorocarbon chain is in a liquid state in low temperature, i.e. liquid fluorocarbon, when ambient pressure reduction or temperature are increased to its boiling
When point, liquid-gas phase transition can occur, gas is become by liquid.Wherein, the boiling point of perflenapent under normal pressure is 29 DEG C, and human body
Temperature is 37 DEG C, therefore it is a kind of fluorocarbon material for being well suited to preparation solution gas phase modification acoustic contrast agent.
Summary of the invention
It is an object of the invention to there are problem and shortage for above-mentioned, a kind of package liquid fluorocarbon, preparation process are provided
Easy, safe and non-toxic, cheap, good biocompatibility the preparation side based on chitosan derivatives nanoscale ultrasound contrast agents
Method.
The technical scheme of the present invention is realized as follows:
Preparation method of the present invention based on chitosan derivatives nanoscale ultrasound contrast agents, the contrast agent is by liquid fluorine
Carbon kernel and chitosan derivatives shell are constituted, and the material of the chitosan derivatives shell is positive hexanoyl carboxymethyl chitosan,
Its main feature is that the following steps are included:
(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 processing, makes liquid
Fluorine carbon is wrapped in positive hexanoyl carboxymethyl chitosan and receives to get to what is be made of liquid fluorocarbon kernel and chitosan derivatives shell
Meter level acoustic contrast agent.
Wherein, the specific operation method is as follows for the step (1):
(11) 250mg carboxymethyl chitosan is weighed, after being dissolved in 20ml pure water and stirring for 24 hours, 20ml methanol is added
It stirs evenly；
(12) n-caproic anhydride of 0.33ml is added dropwise under quick magnetic agitation, and maintains revolving speed stirring in 1000rpm
12 h are reacted under the conditions of mixing；
(13) for 24 hours with ethanol water (25%, v/v) dialysis, then with pure water dialysis for 24 hours, freeze-drying is modified
Chitosan derivatives afterwards --- positive hexanoyl carboxymethyl chitosan, and pass through FTIR spectrum and proton magnetic wave spectrum analysis
Identify the product.
The specific operation method is as follows for the step (2):
It takes positive hexanoyl carboxymethyl chitosan 20mg~50mg to be scattered in 10ml pure water to dissolve.
The specific operation method is as follows for the step (3):
(31) after taking positive hexanoyl carboxymethyl chitosan sugar aqueous solution, ice bath to handle 10min, 100ul~200ul liquid fluorine is added
Carbon；
(32) under condition of ice bath, ultrasound cream is carried out to the positive hexanoyl carboxymethyl chitosan sugar aqueous solution that joined liquid fluorocarbon
Change processing to get chitosan derivatives nanoscale ultrasound contrast agents are arrived, and contrast agent is placed in spare in 4 DEG C of refrigerators；It is described super
Sound meets following condition: ultrasonic amplitude 20%, total sonication time 2min, every ultrasound starting 3s, intermittent time 1s.
The material of the liquid fluorocarbon kernel is perflenapent or perflexane.
Compared with prior art, the present invention having the advantage that
Material used in the present invention be by natural polymers it is chitin modified after it is obtained, material is safe and non-toxic,
Biocompatibility is good, price is lower than synthetic phospholipid；Moreover, preparation process of the invention is easy, have a wide range of application, can by pair
Polymer is modified, and is made it have targeting characteristic, is prepared into targeted ultrasound contrast agent；Meanwhile the polymer can be used as medicine
Object and genophore.
The present invention will be further described below with reference to the drawings.
Detailed description of the invention
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.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below.
Embodiment 1: synthesis chitosan derivatives --- positive hexanoyl carboxymethyl chitosan
250 mg carboxymethyl chitosans are weighed, the molecular weight of the carboxymethyl chitosan is 20 KD, and it is pure to be dissolved in 20ml
Water and stir 24 h after, be added 20ml methanol stir evenly；Then 0.33 ml is being added dropwise under the conditions of quick magnetic agitation just
Caproic anhydride, and 12h is stirred to react under 1000 rpm revolving speeds；Then 24 h of ethanol water (25%, v/v) dialysis are used, then are used
Pure water 24 h of dialysis, freeze-drying obtain modified chitosan derivatives --- positive hexanoyl carboxymethyl chitosan, and pass through
FTIR spectrum and proton magnetic wave spectrum analysis identify the product.
The infrared spectrum of carboxymethyl chitosan and positive hexanoyl carboxymethyl chitosan is as shown in Figure 1, due to carboxymethyl chitosan
Deacetylation is high, so corresponding to the bending vibration of N-H on primary amino group, covers the absorption peak of amide I band and II band.Product exists
1500-1550cm-1There is absorption peak, and bands of a spectrum are remarkably reinforced, 2960cm-1And 2870cm-1Belong to the stretching vibration of methyl,
Absorption peak enhancing, illustrates that carbochain is already connected on amino.
The proton magnetic wave spectrogram of positive hexanoyl carboxymethyl chitosan is as shown in Fig. 2, it is second that its chemical shift, which is 1.9-2.9ppm,
Acylate group, chemical shift are that 3.6-4.0ppm is hydrogen position in saccharide ring, and 2.0 ppm are carboxymethyl chitosan-COCH3On
Hydrogen.Chemical shift 0.8ppm, 1.2 ppm, 1.5 ppm, 2.2ppm are to be connected on amino methyl and methylene on hexane.
Embodiment 2: the cytotoxicity experiment of positive hexanoyl carboxymethyl chitosan
Poison of the positive hexanoyl carboxymethyl chitosan material to A2780 cell of various concentration (1mg, 5mg, 10mg) is respectively set
Property effect.Positive hexanoyl carboxymethyl chitosan material is dispersed with the culture medium containing 10% serum, is configured to concentration difference 1
Mg/ml, 5 mg/ml, 10 mg/ml；It is recovered and is passed on when it is to exponential phase of growth to A2780 cell, carry out cytometer
Number, adjusts cell density as needed；With 96 orifice plate cover plant cells, every hole cell suspension 100ul, every hole is inoculated with 1*10 respectively4
A cell is cultivated for 24 hours in 37 DEG C of incubators；Plate inner cell is cleaned with PBS, the positive hexanoyl of 3 kinds of various concentrations is added
Carboxymethyl chitosan material, every kind of concentration are arranged 6 multiple holes, and control group is added the blank cultures for being free of nanoparticle, cell with
Nanoparticle is incubated for 24 h；The culture medium on the former culture plate of 10% CCK-8 culture medium absorption is configured, the CCK- containing 10% is added
8, to the culture medium containing 10% serum, put back to incubator and continue to cultivate 1h.Extinction at 450 nm is measured in full-automatic microplate reader
Spend A.It is calculated according to formula, cell survival rate=[ (experimental group OD value-zeroing group OD value)/(control group OD value-zeroing
Group OD value) ] × 100%.
Fig. 3 is as the result is shown: as material concentration increases, the cytotoxicity of positive hexanoyl carboxymethyl chitosan is increased with it, and is deposited
Motility rate reduces, but when increasing to 10mg/ml, cell survival rate still 90% or more, illustrate the biological safety of the material compared with
It is good, no obvious vitro cytotoxicity effect.
Embodiment 3: the nanoscale ultrasound contrast agents of positive hexanoyl carboxymethyl chitosan package liquid fluorocarbon are prepared, step is such as
Under:
(1) chitosan derivatives are synthesized --- positive hexanoyl carboxymethyl chitosan；
(2) configure positive hexanoyl carboxymethyl chitosan sugar aqueous solution, method is: take positive 20 mg of hexanoyl carboxymethyl chitosan~
50 mg are scattered in dissolution in 10 ml pure water and are formed as chitosan derivatives micellar aqueous solution, and chitosan derivatives micella is water-soluble
The concentration of liquid is 2 mg/ml；
(3) after taking positive hexanoyl carboxymethyl chitosan sugar aqueous solution ice bath to handle 10 min, 100 ul~200ul liquid fluorine is added
Carbon, the liquid fluorocarbon are perflenapent or perflexane, and in the present embodiment, liquid fluorocarbon is perflenapent, and just oneself
The ratio of acyl carboxymethyl chitosan and perflenapent is 1mg:10 μ l；Then under condition of ice bath, to the shell that liquid fluorocarbon is added
Polysaccharid derivative micellar aqueous solution carries out ultrasonic emulsification processing, is wrapped in liquid fluorocarbon in positive hexanoyl carboxymethyl chitosan, i.e.,
Chitosan derivatives nanoscale ultrasound contrast agents are obtained, are placed in spare in 4 DEG C of refrigerators；
The ultrasound meets following condition: ultrasonic amplitude 20%, and total ultrasonic time is 2min, ultrasound starting 3s, interval
Time 1s.
Embodiment 4: the characterization of nanoscale ultrasound contrast agents
(1) surface topography is carried out using optical microscopy and transmission electron microscope and constitutes observation
After being diluted nanoscale ultrasound contrast agents with physiological saline, is observed under inverted microscope, it is heated to
37 DEG C, surface glossy clear good to contrast agent droplet distribution can be observed, size is more uniform.Preparation is observed under transmission electron microscope
Obtained acoustic contrast agent is rounded, and surface is smooth bright, as shown in Figure 4.
(2) partial size and current potential:
The nanometer acoustic contrast agent of package liquid fluorocarbon obtained is surveyed after taking ultrapure water to dilute with dynamic light scattering method
Its fixed partial size simultaneously detects its Zeta potential using laser particle analyzer, its average grain diameter is (205.9 ± 45.31) nm as the result is shown,
As shown in figure 5, 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 perflenapent is wrapped up
37 DEG C of de aerated waters are filled in the sink, extract the acoustic contrast agent suspension of 4 ml to Sparta's suction pipe with syringe,
It is placed under de aerated water and preheats 5 min, ultrasonic probe is immersed in underwater and face Sparta suction pipe, with Philips IU-22
Type diasonograph (probe model L12-5) carries out ultrasonic imaging, is respectively compared in different carriers concentration (2mg-5mg), difference
Under PFP volume ratio (1-2%), different time sections (0-12min) and same concentration, seen at different mechanical indexes (MI=0.4-0.6)
Examine its imaging effect.It can be seen that Echoenhance in Sparta's suction pipe, at intensive choice refreshments shape echogram, as shown in Figure 7.
In addition, being found in chitosan derivatives nanoscale ultrasound contrast agents prepared by the present invention: changing chitosan and liquid
Ratio, ultrasonic power and time of fluorine carbon etc. can all influence the yield, particle diameter distribution and contrasting effects of contrast agent.
Moreover, the present invention is to be prepared for having amphiphilic chitosan derivatives micella water using nanoparticle self assembly mode
Solution (positive hexanoyl carboxymethyl chitosan sugar aqueous solution), with this there is amphiphilic chitosan to spread out liquid fluorocarbon under cryogenic
Biological micellar aqueous solution mixing, is emulsified using ultrasonic cell disintegration instrument or supersonic wave cleaning machine, is successfully prepared with amphiphilic
Property chitosan derivatives --- positive hexanoyl carboxymethyl chitosan is shell, wrap up the nanoscale ultrasound contrast agents of liquid fluorocarbon.
The present invention is to be described by case study on implementation, but do not limit the invention, reference description of the invention,
Other variations of the disclosed embodiments, are such as readily apparent that the professional person of this field, such variation should belong to
Within the scope of the claims in the present invention restriction.
Claims (2)
1. a kind of preparation method based on chitosan derivatives nanoscale ultrasound contrast agents, the contrast agent by liquid fluorocarbon kernel and
Chitosan derivatives shell is constituted, and the material of the chitosan derivatives shell is positive hexanoyl carboxymethyl chitosan, and feature exists
In the following steps are included:
(1) positive hexanoyl carboxymethyl chitosan is synthesized；
(2) positive hexanoyl carboxymethyl chitosan sugar aqueous solution is prepared；
(3) liquid fluorocarbon is added drop-wise in positive hexanoyl carboxymethyl chitosan sugar aqueous solution, carries out ultrasonic emulsification processing, makes liquid fluorocarbon
It is wrapped in positive hexanoyl carboxymethyl chitosan to get to the nanoscale being made of liquid fluorocarbon kernel and chitosan derivatives shell
Acoustic contrast agent；
The specific operation method is as follows for the step (1):
(11) 250mg carboxymethyl chitosan is weighed, after being dissolved in 20ml pure water and stirring for 24 hours, the stirring of 20ml methanol is added
Uniformly；
(12) n-caproic anhydride of 0.33ml is added dropwise under quick magnetic agitation, and maintains revolving speed in the stirring bar of 1000rpm
12h is reacted under part；
(13) for 24 hours, then for 24 hours with pure water dialysis with the ethanol water dialysis of 25%v/v, freeze-drying obtains modified shell
Polysaccharid derivative --- positive hexanoyl carboxymethyl chitosan, and should by FTIR spectrum and the identification of proton magnetic wave spectrum analysis
Product；
The specific operation method is as follows for the step (2):
It takes positive hexanoyl carboxymethyl chitosan 20mg~50mg to be scattered in 10ml pure water to dissolve；
The specific operation method is as follows for the step (3):
(31) after taking positive hexanoyl carboxymethyl chitosan sugar aqueous solution, ice bath to handle 10min, 100ul~200ul liquid fluorocarbon is added；
(32) under condition of ice bath, the positive hexanoyl carboxymethyl chitosan sugar aqueous solution that joined liquid fluorocarbon is carried out at ultrasonic emulsification
Contrast agent is placed in spare in 4 DEG C of refrigerators to get arriving chitosan derivatives nanoscale ultrasound contrast agents by reason；The ultrasound is full
The following condition of foot: ultrasonic amplitude 20%, total sonication time 2min, every ultrasound starting 3s, intermittent time 1s.
2. the preparation method according to claim 1 based on chitosan derivatives nanoscale ultrasound contrast agents, it is characterised in that:
The material of the liquid fluorocarbon kernel is perflenapent or perflexane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610693711.0A CN106139174B (en) | 2016-08-22 | 2016-08-22 | A kind of preparation method based on chitosan derivatives nanoscale ultrasound contrast agents |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610693711.0A CN106139174B (en) | 2016-08-22 | 2016-08-22 | A kind of preparation method based on chitosan derivatives nanoscale ultrasound contrast agents |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106139174A CN106139174A (en) | 2016-11-23 |
| CN106139174B true CN106139174B (en) | 2019-08-09 |
Family
ID=57341955
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610693711.0A Active CN106139174B (en) | 2016-08-22 | 2016-08-22 | A kind of preparation method based on chitosan derivatives nanoscale ultrasound contrast agents |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106139174B (en) |
Families Citing this family (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)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104524602A (en) * | 2014-12-26 | 2015-04-22 | 宁波市第一医院 | Folate receptor targeting ultrasound contrast nanobubble and preparation method thereof |
-
2016
- 2016-08-22 CN CN201610693711.0A patent/CN106139174B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104524602A (en) * | 2014-12-26 | 2015-04-22 | 宁波市第一医院 | Folate receptor targeting ultrasound contrast nanobubble and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Self-Assembled Hollow Nanocapsule from Amphiphatic Carboxymethyl-hexanoyl Chitosan as Drug Carrier;Kun-Ho Liu et al.;《Macromolecules》;20080808;第41卷;第6511-6516页 * |
| 构建新型液态氟碳壳聚糖纳米超声造影微粒;陈玲等;《中国医学影像技术》;20131231;第29卷(第4期);第493-497页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106139174A (en) | 2016-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Functionalized holmium‐doped hollow silica nanospheres for combined sonodynamic and hypoxia‐activated therapy | |
| Brismar et al. | Magnetite nanoparticles can be coupled to microbubbles to support multimodal imaging | |
| CN102036652B (en) | Be suitable for the cholesteric nanostructured of chelating | |
| CN105997879B (en) | A kind of nano vesicle and its preparation method and application of pH and temperature doubling sensitivity | |
| Kim et al. | Nanosized ultrasound enhanced-contrast agent for in vivo tumor imaging via intravenous injection | |
| US8940277B2 (en) | Intracellular microbubble for imaging an anatomical site | |
| Slagle et al. | Click conjugation of cloaked peptide ligands to microbubbles | |
| Li et al. | A preliminary study of photoacoustic/ultrasound dual-mode imaging in melanoma using MAGE-targeted gold nanoparticles | |
| Mannaris et al. | Acoustically responsive polydopamine nanodroplets: a novel theranostic agent | |
| Nakatsuka et al. | DNA-Coated Microbubbles with Biochemically-Tunable Ultrasound Contrast Activity | |
| CN109260480A (en) | A kind of chitosan nano meter level acoustic contrast agent and the preparation method and application thereof carrying adriamycin | |
| CN106581698A (en) | Preparation method for ultrasonic fluorescence bimodal nano-probe for recognizing unstable plaque of atherosclerosis | |
| CN111632154A (en) | Phase-transition nanobubble, preparation method and application thereof | |
| CN106139174B (en) | A kind of preparation method based on chitosan derivatives nanoscale ultrasound contrast agents | |
| CN104524602B (en) | Folacin receptor targeted ultrasound contrast nanometer microvesicle | |
| TWI515009B (en) | Photosensitizer particles for medical imaging and/or photodynamic therapy | |
| CN111317817B (en) | Targeting photoacoustic imaging nano molecular probe and application thereof | |
| Osborn et al. | Echogenic exosomes as ultrasound contrast agents | |
| CN100562341C (en) | The application of cell nucleus targeting chitosan-fatty acid graft as medicine carrier micelle | |
| CN103638534B (en) | A kind of nano-lipid acoustic contrast agent and preparation method | |
| CN104258391A (en) | Multi-functional stimuli sensitive polymer-gold nanocage carrier and preparation method thereof | |
| Lv et al. | Multifunctional nanosnowflakes for T1-T2 double-contrast enhanced MRI and PAI guided oxygen self-supplementing effective anti-tumor therapy | |
| Cheng et al. | Covalently conjugated hydrogelators for imaging and therapeutic applications | |
| CN104587497B (en) | The preparation method of folacin receptor targeted ultrasound contrast nanometer microvesicle | |
| CN111420073A (en) | AMD070 and ICG carrying multi-modal targeting nanobubble and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |