CN105536000B - A kind of acoustic contrast agent and its preparation method and application based on pentaerythritol ester - Google Patents
A kind of acoustic contrast agent and its preparation method and application based on pentaerythritol ester Download PDFInfo
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- CN105536000B CN105536000B CN201510917095.8A CN201510917095A CN105536000B CN 105536000 B CN105536000 B CN 105536000B CN 201510917095 A CN201510917095 A CN 201510917095A CN 105536000 B CN105536000 B CN 105536000B
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- contrast agent
- acoustic contrast
- acoustic
- pentaerythritol ester
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- 230000002093 peripheral effect Effects 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 235000010988 polyoxyethylene sorbitan tristearate Nutrition 0.000 description 1
- 239000001816 polyoxyethylene sorbitan tristearate Substances 0.000 description 1
- 229940099511 polysorbate 65 Drugs 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000004088 pulmonary circulation Effects 0.000 description 1
- 230000000191 radiation effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 210000002268 wool Anatomy 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
- A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
-
- 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
- A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
Landscapes
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Acoustics & Sound (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Radiology & Medical Imaging (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention relates to field of medicaments, specifically disclose a kind of acoustic contrast agent and its preparation method and application based on pentaerythritol ester.The coated fertilizer of acoustic contrast agent of the present invention includes at least a kind of pentaerythritol ester.Acoustic contrast agent coating of the present invention can also form the acoustic contrast agent of microbubble structure by the filling gas into coating or filling liquid body comprising components such as surfactant, high molecular polymer and phospholipids compounds.The preparation of this acoustic contrast agent uses sound and vibration cavitation method, and preparation process is simple, controllable；The lot of advantages such as prepared acoustic contrast agent biocompatibility is good, the effective time with good acoustic response ability, development is long, low in cost and suitable industrialized production, and can be used as drug and genophore, with wide potential applicability in clinical practice.
Description
Technical field
The present invention relates to field of medicaments, specifically disclose a kind of based on the acoustic contrast agent of pentaerythritol ester and its preparation side
Method and purposes.
Background technique
Ultrasonic imaging technique refers to, based on the difference of human tissue organ's acoustic properties, using ultrasonic wave in difference
The strong and weak difference of reflection and scattered signal in tissue comes response organization's internal physiological and pathological information, to reach the mesh of diagnosis
's.Compared with other imaging methods, ultrasonic examination is "dead" without any pain, does not need special environmental condition, provides
Information it is accurate, abundant, intuitive, will be widely welcomed by patient and doctor.Ultrasound medicine develops very fast, short decades
Just become clinical indispensable routine inspection means.Due to the economic and practical of ultrasonic examination, diasonograph is in China
Popularity is significantly larger than the image technology of the valuableness such as CT, magnetic resonance.
Acoustic contrast agent (ultrasoundcontrastagentsUCAs) is by changing the ultrasonic characteristic of tissue (as backwards
Scattering coefficient, attenuation coefficient, the velocity of sound and nonlinear effect etc.) to make the echo signal at place position significantly increase, it can obtain
To more abundant information with auxiliary diagnosis.Acoustic contrast agent is in human body tiny blood vessels and tissue blood flow's perfusion detection and imaging side
Mask has great advantage.Theoretically, acoustic contrast agent can be used for any organ blood flow filling in addition to bone and lung in vivo
The evaluation of note.The development of acoustic contrast agent had gone through for two generations：First generation contrast agent is to wrap up one layer in air bubble periphery
As putamina, stability is good for albumin, lipid, polymer or surfactant, size it is small (<7 μm), therefore microvesicle is infused through vein
After penetrating, left ventricle and peripheral blood vessel are reached by pulmonary circulation and, because reinforcing effect is faint, the duration is short, examined to make its development
Disconnected value less, has been gradually backed out contrast agent market and research field after 2000；The microvesicle radiography of second generation fluoro-gas
Agent, the molecular weight of fluorocarbon gas bigger than air have lower solubility and the spread more, are not easy in microvesicle through steep that wall
Diffusion, while it is inert gas, can keep good stability in vivo, therefore the second generation microbubble contrast agent prepared is not only
Reinforcing effect is significant, and the duration is obviously prolonged, and has specific diagnostic value for a variety of diseases.
The important development direction of acoustic contrast agent development is the improvement of filmogen, it is therefore an objective to use contrast agent and more pacify
Entirely, the radiography duration is longer.Filmogen mainly includes following some protein class, phospholipid, high molecular polymerization species, table
Face activating agent class.Since lipid material has good biological safety, and adipose membrane contrast agent is made of macromolecular lipid
Adipose membrane it is more more stable than the film that albuminate is formed, more flexible, contrasting effects are more preferable, and the duration is longer, are more able to satisfy
Clinical needs.Therefore, the contrast agent of new approval listing, D efinity and Imageat, the SonoVue in Europe such as the U.S. are equal
Belong to adipose membrane contrast agent, wherein SonoVue has been approved by carries out clinical use at home.But the short radiography duration is always to perplex
The problem of acoustic contrast agent development person, and influence the major reason that contrast agent is universal and further develops.According to text
Report is offered, for SonoVue to the effective Enhanced time of parenchymatous organ only 4~8 minutes, it is ultrasonic in detail that this is difficult one internal organs of satisfaction
Check the needs of (generally requiring 10~15 minutes).From the point of view of development, acoustic contrast agent will be controlled further in intervention from now on
The development for the treatment of field (not only diagnoses), and the microbubble contrast agent targeted release of gene or therapeutic agent is such as carried using ultrasound destruction
Gene transfer or drug targeting treatment are mediated, just with greater need for stability of the contrast agent in blood circulation is improved, extension is followed in body
The ring time.And the price of SonoVue is 110 dollars/bottle, it is expensive.Therefore the side of the development of third generation acoustic contrast agent
To must be further increase the radiography duration and reduce preparation cost.
In view of this, the present invention is specifically proposed.
Summary of the invention
The object of the present invention is to provide a kind of preparation cost is cheap, organize in developing time it is long, based on pentaerythritol ester
Acoustic contrast agent and its preparation method and application.
The present invention provides a kind of acoustic contrast agent based on pentaerythritol ester, and the acoustic contrast agent contains coated fertilizer,
The coated fertilizer contains one or more pentaerythritol esters.
Pentaerythritol ester of the present invention preferably has following general formula of the chemical structure：
Wherein：Y=1~21；X represents any group that can connect pentaerythrite skeleton and hydrophobic fat chain.
Pentaerythritol ester of the present invention is total to by hydrophilic quaternary the eleventh of the twelve Earthly Branches tetrol head base and fatty last-of-chain base and linking group
Valence is formed by connecting, and chemical property is stablized.
The pentaerythritol ester of the present invention is preferably：
Wherein, y=10~16.
Pentaerythritol ester of the present invention is most preferably pentaerythritol monostearate, pentaerythrite list palmitate.
The coated fertilizer of acoustic contrast agent of the present invention contains following components：In terms of mass parts, pentaerythritol ester 0.5~
2.5 parts, 0~2.0 part of surfactant, 0.6~2.0 part of high molecular polymer, 0~2.0 part of phospholipids compounds.
Coating of the present invention has good contrast enhancing effects, and chemical property is stablized, and shelf lives are long；Coating of the present invention
High resilience, pressure resistance is functional in blood circulation, and the radiography duration is longer；Coating of the present invention has good in vivo
Good degradability, has no toxic and side effect to human body, use is safe, is able to satisfy clinical medical needs.
Surfactant of the present invention be preferably span 20, span 40, sorbester p18, sorbester p38, sorbester p17, span 85,
Tween 21, polysorbate40, polysorbate60, Tween61, polysorbate65, Tween 80, sorbimacrogol oleate100, polysorbate85 and cetyl trimethyl bromination
One of ammonium is a variety of.
The phospholipids compounds are preferably lecithin, hydrogenated soya phosphatide, hydrogenation yolk phospholipid, two palmityl phosphatidyls
Ethyl alcohol, dipalmitoylphosphatidylcholine, dioleoyl phosphatidyl ethanolamine, polyethylene glycol-distearoyl phosphatidyl ethanolamine, 1,2-
Distearyl acyl group-sn- glyceryl -3- phosphatidyl choline, 1, bis- palmityl-sn- glyceryl -3- phosphatidyl choline of 2- and phosphatide
One of acyl serine is a variety of.
The high molecular polymer is polyethylene glycol mono fatty acid ester and/or polyethylene glycol double acid ester, is preferably gathered
Ethylene glycol fatty acid ester, further preferably relative molecular mass be 1 000~7000 polyethylene glycol mono fatty acid ester and/
Or polyethylene glycol double acid ester.
The present invention provides a kind of preferred coated fertilizer, in terms of mass parts, containing pentaerythritol monostearate 0.5~
2.5 parts, 0.6~2.0 part of polyethylene glycol mono stearate.
The present invention provides a kind of preferred coated fertilizer, in terms of mass parts, contains pentaerythritol monostearate 1.77
Part, 1 part of polyethylene glycol mono stearate.
It is prepared into acoustic contrast agent with such coated fertilizer, effective Enhanced time is 13min-1 5min, development effect
Well.
Preferably, acoustic contrast agent of the present invention further includes filling gas or filling liquid.
Wherein, the filling gas is carbon dioxide, in oxygen, air, nitrogen, inert gas, sulfur fluoride, fluorocarbon gas
One or more, preferably perfluoropropane or sulfur hexafluoride；
The filling liquid is one of perfluoro bromide octane, perfluorooctane, perflexane, perflenapent or a variety of.
Filling gas selected by the present invention or liquid are usually that high density inert gas or liquid (are not readily dissolved in water or blood
Liquid), such material and corresponding membrane material can form thin and pliable microvesicle, and the stable time is long, and vibration and echoing characteristics are good.
The present invention provides a kind of methods for preparing the acoustic contrast agent, comprise the steps of：
Step (1)：It into the coated fertilizer containing pentaerythritol ester, is added phosphate buffer solution (PBS), mixing is equal
It is even, produce coating solution；
Step (2)：It is passed through filling gas into the coating solution or filling liquid is added dropwise, ultrasonic treatment is until obtain milky white
Color suspension；Milky suspension is stood to being divided into three layers, middle layer microvesicle is taken and is rinsed repeatedly with PBS buffer solution to lower layer's solution
Clarification, obtains acoustic contrast agent.
The ultrasonic treatment is preferably carried out using Ultrasonic Cell Disruptor, and the frequency of the preferably described ultrasonic treatment is 20~25kHz,
Power is 150~600W.
Coating solution is preferably made in the following manner in step (1) of the present invention：In terms of mass parts, by pentaerythritol ester
0.5~2.5 part, 0~2.0 part of surfactant, 0.6~2.0 part of high molecular polymer, 0~2.0 part of phospholipids compounds plus
Enter into organic solvent to being completely dissolved, PBS buffer solution, water bath sonicator point is added in rotary evaporation to organic solvent evaporating completely
It dissipates.
Wherein, the organic solvent is preferably chloroform.
In addition, the coating solution of step (1) of the present invention can also be made in the following manner：In terms of mass parts, by Ji Wusi
0.5~2.5 part of alcohol ester, 0~2.0 part of surfactant, 0.6~2.0 part of high molecular polymer, 0~2.0 part of phospholipids compounds
Grinding uniformly, is then dispersed in PB S, stirs evenly, obtain suspension；By the suspension at 100~140 DEG C of high temperature, high pressure
Sterilize 10-15min under the conditions of 0.1~0.3MPa, continuously stirs, is cooled to room temperature to get coating solution after sterilizing.
The present invention prepares contrast agent using sound and vibration cavitation, low in cost, manufacture craft is simply controllable, and it is big to be conducive to industrialization
Large-scale production.
Acoustic contrast agent of the present invention can save in the following way：The acoustic contrast agent is freeze-dried, is filled with two
One of carbonoxide, oxygen, air, nitrogen, inert gas, sulfur fluoride, fluorocarbon gas or a variety of preservations.
It is preferred that appropriate skeleton supporting agent, stabilizer and/or surface-active is added before the acoustic contrast agent freeze-drying
Agent.It is described appropriate preferably living according to every 10ml acoustic contrast agent addition 0.3~3g skeleton supporting agent, stabilizer and/or surface
The combined ratio of one or more of property agent.
Wherein, the skeleton supporting agent is preferably one of poloxamer, polyethylene glycol, sucrose, chitosan, trehalose
Or several combination；Stabilizer is preferably：One of mannitol, sorbierite, glucose, lactose, galactolipin, palmitinic acid are several
The combination of kind；Surfactant is preferably：One of polyvinyl pyrrolidone, polysorbate60, Tween 80, sorbester p18, sorbester p17 or
Several combinations.Before acoustic contrast agent is freeze-dried by the present invention, it is living that appropriate skeleton supporting agent, stabilizer and/or surface is added
Property agent, advantage are that they are both effective cryoprotective agent and good freeze drying protectant, can get uniform one after freeze-drying
It causes, surface is smooth, microbubble contrast agent that is stable, being easy to aquation dispersion.Acoustic contrast agent of the present invention is in ultrasonic imaging, targeting
There is good application in drug and genophore.
The physiology of certain volume is added into the acoustic contrast agent freeze-dried powder before every time using ultrasonic contrast by the present invention
Salt water shakes up.
Microvesicle of the present invention generates cavitation effect as cavitation nucleus, by energy radiation effects, makes to discharge after microbubble destruction
Drug is able to enter vascular wall even tissue space.Therefore, contrast agent of the present invention can carry certain certain drugs, to play
Targeted therapy effect.
Acoustic contrast agent prepared by the present invention based on pentaerythritol ester has the advantage that：1, biocompatibility is good
It is good；2, membrane structure is stablized；3, preparation process is simply controllable；4, there is good acoustic response ability；5, ultrasound enhancing is developed
Effective time is long；6, have a wide range of application, can be used as drug and genophore；7, acoustic contrast agent preparation cost of the present invention is cheap,
It is suitble to industrialized production.
Number of the present invention is mass parts, and unit is g or kg, depending on the circumstances；The phosphate radical of PBS buffer solution of the present invention
Molar concentration is 10mmol/L, pH=7.2~7.4.
Detailed description of the invention
Fig. 1 is image of the microbubble contrast agent under 400 times of optical microscopies；
Fig. 2 is ultrasonic contrast result figure after microbubble contrast agent and physiological saline injection medical silicone tube, wherein a and b difference
It is the ultrasonic development of the longitudinal section and cross section after silicone tube saline injection as a result, c and d are silicone tube injection ultrasound respectively
The ultrasonic development result of longitudinal section and cross section after contrast agent；
Fig. 3 is the ultrasonic development result figure of new zealand white rabbit kidney, wherein figure a is the kidney injected before acoustic contrast agent
Developed image, figure b are the kidney developed image after acoustic contrast agent 3min.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
Embodiment 1：The preparation of acoustic contrast agent
The present embodiment provides a kind of coating microcapsular ultrasound contrast agent based on pentaerythritol ester, specifically comprises the steps of：
Step (1)：Pentaerythritol monostearate 1.77g, polyethylene glycol mono stearate 1g and NaCl1.50g are taken, is set
It is fully ground, is scattered in 50mLPBS in mortar, magnetic agitation mixes to obtain suspension；The suspension is placed in 120 DEG C to go out
Sterilize under the conditions of pressure 0.2MPa 12min in bacterium pot, continuously stirs, is cooled to room temperature later, obtains coating solution；
Step (2)：Perfluoropropane (C is passed through into the coating solution3F8) gas, with So nicator4000 ultrasonic wave
Pop one's head in the continuous ultrasound 3min under peak swing for " 1/2 " of broken instrument, obtains uniform milky suspension, is collected in
35min is stood in 250mL separatory funnel, various sizes of bubble is divided into apparent three layers, in collection due to natural buoyancy effect
Interbed microvesicle is simultaneously rinsed with PBS buffer solution until the clarification of lower layer's solution, obtains acoustic contrast agent repeatedly；
Step (3)：The acoustic contrast agent is collected in cillin bottle, is filled with C after mixing in equal volume with PBS3F8Gas,
Sealing, is placed in 4 DEG C of refrigerators and saves.
The effective Enhanced time of the present embodiment acoustic contrast agent is 15min, and development effect is good.
Embodiment 2：The preparation of acoustic contrast agent
The present embodiment provides a kind of coating microcapsular ultrasound contrast agent based on pentaerythritol ester, specifically comprises the steps of：
Step (1)：In terms of mass parts, by pentaerythrite monostearate 1.77g, polyethylene glycol mono stearate 1g and
NaCl1.5g is added in the chloroform of 20mL to being completely dissolved, then 50mLPBS buffer is added to remove chloroform in rotary evaporation,
Water bath sonicator dispersion, obtains coating solution；
Step (2)：SF is passed through into the coating solution6Gas, with " the 1/ of Sonicator4000 sonicator
2 " probes continuous ultrasound under peak swing handles 4min, obtains uniform milky suspension, is collected in 250mL liquid separation leakage
After standing 30min in bucket, various sizes of bubble collects middle layer microvesicle simultaneously since natural buoyancy effect is divided into apparent three layers
It is rinsed repeatedly with P BS buffer solution until the clarification of lower layer's solution, obtains acoustic contrast agent；
Step (3)：Acoustic contrast agent is collected in cillin bottle, is filled with SF after mixing in equal volume with PBS6Gas, sealing,
It is placed in 4 DEG C of refrigerators and saves.
The effective Enhanced time of the present embodiment acoustic contrast agent is 13min, and development effect is good.
Embodiment 3：The preparation of acoustic contrast agent
The present embodiment provides a kind of coating microcapsular ultrasound contrast agent based on pentaerythritol ester, specifically comprises the steps of：
Step (1)：Take pentaerythrite list palmitate 0.855g, 60 1.5g of surface active agent tween, high molecular polymer
PBS50mL is added in polyethylene glycol double acid ester 1g, phospholipids compounds lecithin 1.73g, is uniformly mixed, it is molten to produce coating
Liquid；
Step (2)：SF is passed through into the coating solution6Gas, with " the 1/ of Sonicator4000 sonicator
2 " probes continuous ultrasound under peak swing handles 4min, obtains milky suspension；It is collected in 250mL separatory funnel and is stood
After 30min, various sizes of bubble is collected middle layer microvesicle and is delayed with PBS since natural buoyancy effect is divided into apparent three layers
Solution is rushed to be rinsed repeatedly until the clarification of lower layer's solution, obtains acoustic contrast agent；
Step (3)：Acoustic contrast agent is collected in cillin bottle, is filled with SF after mixing in equal volume with PBS6Gas, sealing,
It is placed in 4 DEG C of refrigerators and saves.
The effective Enhanced time of the present embodiment acoustic contrast agent is 14min, and development effect is good.
Embodiment 4：The preparation of acoustic contrast agent
The present embodiment provides a kind of coating microcapsular ultrasound contrast agent based on pentaerythritol ester, specifically comprises the steps of：
Step (1)：Take pentaerythritol stearate 1.53g, surfactant sorbester p18 0.984g, high molecular polymerization
PBS50mL, mixing is added in object polyethylene glycol double acid ester 1g, phospholipids compounds dipalmitoylphosphatidylcholine 1.677g
Uniformly, coating solution is produced；
Step (2)：Perfluoropropane (C is passed through into the coating solution3F8) gas, with So nicator4000 ultrasonic wave
Pop one's head in the continuous ultrasound processing 3mi n under peak swing for " 1/2 " of broken instrument, obtains uniform milky suspension, is collected
35mi n is stood in 250mL separatory funnel, various sizes of bubble is received since natural buoyancy effect is divided into apparent three layers
Collection middle layer microvesicle is simultaneously rinsed with PBS buffer solution until the clarification of lower layer's solution, obtains acoustic contrast agent repeatedly；
Step (3)：The acoustic contrast agent is collected in cillin bottle, is filled with C after mixing in equal volume with PBS3F8Gas,
Sealing, is placed in 4 DEG C of refrigerators and saves.
The effective Enhanced time of the present embodiment acoustic contrast agent is 16min, and development effect is good.
Comparative example 1
A kind of coating microcapsular ultrasound contrast agent for being not based on pentaerythritol ester is present embodiments provided, specifically includes following step
Suddenly：
Step (1)：In terms of mass parts, Span 601.5g, polyethylene glycol mono stearate 0.792g and NaCl1.5g are taken, is set
It is fully ground, is scattered in 50mLPBS in mortar, magnetic agitation mixes.Uniformly mixed suspension is placed in 120 DEG C of sterilizings
Sterilize 12min in pot, continuously stirs, is cooled to room temperature later, obtains coating solution；
Step (2)：Perfluoropropane (C is passed through into the coating solution3F8) gas, with So nicator4000 ultrasonic wave
Pop one's head in the continuous ultrasound processing 3mi n under peak swing for " 1/2 " of broken instrument, obtains uniform milky suspension, is collected
35mi n is stood in 250mL separatory funnel, various sizes of bubble is received since natural buoyancy effect is divided into apparent three layers
Collection middle layer microvesicle is simultaneously rinsed with PBS buffer solution until the clarification of lower layer's solution, obtains acoustic contrast agent repeatedly；
Step (3)：The acoustic contrast agent is collected in cillin bottle, is filled with C after mixing in equal volume with PBS3F8Gas,
Sealing, is placed in 4 DEG C of refrigerators and saves.
Compared with Example 1, difference is only that this comparative example：This comparative example is using Span 60 instead of in embodiment 1
Pentaerythritol monostearate, other conditions are all the same.
The effective Enhanced time of this comparative example acoustic contrast agent is 7min, and development effect is poor.
Embodiment 6：The preparation of freeze-dried powder
2g PLURONICS F87 is added in the acoustic contrast agent 10mL that Example 1 or 2 step of embodiment (2) are prepared,
It is freeze-dried after mixing, temperature is -50 DEG C~-70 DEG C, and vacuum suction pressure is 5~20 × 10-3mtor, time 20
~25h.Aseptically the dried frozen aquatic products being freeze-dried are crushed, is sub-packed in the cillin bottle of 10mL, is passed through perfluoropropane
Gas, capping.
Experimental example 1：The optical microphotograph sem observation of microvesicle
The present embodiment is image of the microvesicle under 400 times of optical microscopies in embodiment 1；Concrete operation step is as follows：With
Isometric PBS dilutes microvesicle suspension, draws 10 μ L microvesicle suspensions drop on clean glass slide with pipettor, carefully closes the lid
Slide is observed size, form and the dispersion of microvesicle using microscope and is taken pictures under different object lens multiples.
Experimental example 2：The partial size and distribution tests of microcapsular ultrasound contrast agent
The size distribution and concentration of novel microvesicle are tested using CoulterMultisizerIII.Operating procedure is as follows：It takes
Freshly prepd 100 μ L of microvesicle suspension is added in 100mLIsotonII dilution, and low speed magnetic agitation is until be uniformly mixed.Again
It is drawn in the IsotonII dilution that 2mL is added to 18mL from the dilution microvesicle suspension of mixing, slight wobble is uniformly mixed.If
Setting analysis volume is 1 00 μ L, tests the particle diameter distribution and concentration of microvesicle.Sample test in triplicate, is averaged.
Experimental example 3：The external supersonic radiography of microbubble contrast agent is tested
De aerated water is filled in the sink, and a transparent medical grade silicone pipe of elasticity (internal diameter about 5mm) is partially disposed in sink
Liquid level is hereinafter, ultrasonic probe is immersed in underwater and face silicone tube.To inject the ultrasonogram of physiological saline as control, with note
Emitter extracts a certain amount of microvesicle suspension and is slowly pushed into from one end of silicone tube, with Pulse inversion harmonic wave mode (Pulse-
InversionHar monicImaging, PIHI) longitudal section of silicone tube and cross section are observed, carry out injection ultrasound
The contrast effect of contrast agent is observed.
Experimental example 4：The internal ultrasonic contrast of microbubble contrast agent is tested
Using new zealand white rabbit as experimental subjects, peripheral vein channel is established through auricular vein in the left ear of rabbit, in conduit
End connects tee tube, and physiological saline is trailed for injecting acoustic contrast agent, a channel in one of channel.New Zealand great Bai
Rabbit and is aided with 0.2% heparin sodium anticoagulation with the 3% sodium intravenous anesthesia (30mg/kgi.p.) of amobarbital.It is complete to rabbit
After anesthesia, it is fixed on rabbit bed with carrying on the back sleeping posture.Right belly wool hair depilatory cream carefully removes, and carries out to rabbit liver and kidney
The ultrasonic examination of contrast agent is not injected, records the image under base state.By a certain concentration doses after image is satisfied
Acoustic contrast agent microvesicle suspension trails 2.0mL normal saline flushing pipeline, in PIHI and energy through rabbit auricular vein bolus at once
Realtime dynamic observation and rabbit liver is recorded and kidney echo is strong under amount doppler mode (PowerDopplerImaging, PDI)
Degree enhancing situation and kidney power doppler blood flow signal enhance situation.Sweep parameter setting, including gain, mechanical index,
Pulse recurrence frequency, scanning plane, scan depths, focal position and visual field etc. remain unchanged in entire angiographic procedure.
Experimental example 5：Preparation loads the acoustic contrast agent of drug
By pentaerythritol monostearate 1.77g, polyethylene glycol mono stearate 1g, NaCl1.5 0g and a certain amount of purple
China fir alcohol is dissolved in the chloroform of 20mL, is then carried out rotary evaporation and is removed chloroform, and it is super that water-bath in 50mLPBS buffer is then added
Sound.C is passed through into obtained suspension3F8Gas is shaken with " 1/2 " probe of Sonicator4000 sonicator in maximum
Continuous ultrasound handles 3min under width.Uniform milky suspension is obtained after sound and vibration, is collected in 250mL separatory funnel and is stood
After 35min, various sizes of bubble is collected middle layer microvesicle and is delayed with PBS since natural buoyancy effect is divided into apparent three layers
Solution is rushed to be rinsed repeatedly until lower layer's solution is clarified.By the microvesicle suspension 10mL of preparation, 2g PLURONICS F87 is added, mixes
After be freeze-dried, temperature be -50~-70 DEG C, vacuum suction pressure be 5~20 × 10-3mtor, the time be 20~25h.
Aseptically the dried frozen aquatic products being freeze-dried are crushed, is sub-packed in the cillin bottle of 10mL, is passed through perfluoropropane gas, are sealed
Lid.
Experimental example 6：Prepare the acoustic contrast agent of carrying gene
Pentaerythritol monostearate 1.239g, cetyl trimethylammonium bromide 0.483g, polyethyleneglycol is stearic
Acid esters 1g and NaCl1.50g, which are placed in mortar, to be fully ground, and is scattered in 50mLPBS, and magnetic agitation mixes.It will be uniformly mixed
Suspension be placed in 120 DEG C of autoclave high temperature high pressure 12min, continuously stir be cooled to room temperature later.Lead into above-mentioned solution
Enter C3F8Gas, with " 1/2 " probe of Sonicator4000 sonicator, continuous ultrasound is handled under peak swing
3min.Uniform milky suspension is obtained after sound and vibration, is collected in 250mL separatory funnel and is stood.After 35min, different rulers
Very little bubble is collected middle layer microvesicle and is rinsed repeatedly with PBS buffer solution since natural buoyancy effect is divided into apparent three layers
Until lower layer's solution is clarified.By the microvesicle suspension 10mL of preparation, 2g PLURONICS F87 is added, is freeze-dried after mixing,
Temperature is -50~-70 DEG C, and vacuum suction pressure is 5~20 × 10-3Mtor, time are 20~25h.It aseptically will be cold
Freeze dried dried frozen aquatic products to crush, is sub-packed in the cillin bottle of 10mL, is passed through perfluoropropane gas, capping.It is incited somebody to action using preceding
The ultrasonic contrast of carrying gene can be obtained in uniformly mixed absorption certain time in solution injection bottle containing siRNA or DNA
Agent.
Although being above described in detail with general explanation, specific embodiment etc. to invention, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.
Claims (8)
1. a kind of acoustic contrast agent based on pentaerythritol ester, which is characterized in that the acoustic contrast agent contains coated fertilizer, institute
The coated fertilizer stated is grouped as by following group, in terms of mass parts, 0.5~2.5 part of pentaerythritol ester, surfactant 0~2.0
Part, 0.6~2.0 part of high molecular polymer, 0~2.0 part of phospholipids compounds and filling gas；
The pentaerythritol ester is pentaerythritol monostearate, pentaerythritol stearate or pentaerythrite list palmitic acid
Ester；
The high molecular polymer is polyethylene glycol mono fatty acid ester or polyethylene glycol double acid ester；
The filling gas is perfluoropropane gas or SF6Gas.
2. acoustic contrast agent as described in claim 1, which is characterized in that the surfactant is span 20, span 40, department
Disk 60, sapn 65, sorbester p17, span 85, tween 21, polysorbate40, polysorbate60, Tween61, tween 65, Tween 80, sorbimacrogol oleate100,
One of polysorbate85 and cetyl trimethylammonium bromide are a variety of；
The phospholipids compounds are lecithin, hydrogenated soya phosphatide, hydrogenation yolk phospholipid, two palmityl phosphatidyl ethanols, two
Palmitoylphosphatidyl choline, dioleoyl phosphatidyl ethanolamine, polyethylene glycol-distearoyl phosphatidyl ethanolamine, 1,2- distearyl
Docosahexaenoyl-sn-glycero -3- phosphatidyl choline, bis- palmityl-sn- glyceryl -3- phosphatidyl choline of 1,2-, phosphatidyl silk ammonia
One of acid is a variety of.
3. a kind of method for preparing acoustic contrast agent as claimed in claim 1 or 2, which is characterized in that comprise the steps of：
Step (1)：Into the coated fertilizer containing pentaerythritol ester, phosphate buffer solution is added, is uniformly mixed, produces coating
Solution；
Step (2)：Filling gas is passed through into the coating solution, ultrasonic treatment is until obtain milky suspension；Milky is hanged
Liquid is stood to being divided into three layers, is taken middle layer microvesicle and is rinsed repeatedly with phosphate buffer to lower layer's solution and clarified, obtains ultrasonic make
Shadow agent.
4. method as claimed in claim 3, which is characterized in that the concrete operations of step (1) are：Coated fertilizer is dissolved in organic
In solvent, organic solvent is removed, phosphate buffer solution is added, ultrasonic disperse is up to the coating solution；
Alternatively, coated fertilizer is dissolved in phosphate buffer, suspension is obtained, by the suspension at 100~140 DEG C, 0.1~
It is sterilized after 10~15min under the conditions of 0.3MPa, is continuously stirring to and is cooled to room temperature up to the coating solution.
5. method as claimed in claim 4, which is characterized in that the organic solvent is chloroform.
6. method as claimed in claim 3, which is characterized in that further include that the acoustic contrast agent of step (2) is prepared into freeze-drying
The step of powder, the step are specially：The acoustic contrast agent is freeze-dried, perfluoropropane gas is then charged with.
7. method as claimed in claim 6, which is characterized in that before the freeze-drying of the step (2) acoustic contrast agent, be added
Appropriate skeleton supporting agent and stabilizer.
8. the method for claim 7, which is characterized in that the skeleton supporting agent is poloxamer, polyethylene glycol, sugarcane
The combination of one or more of sugar, chitosan, trehalose；
The stabilizer is the combination of one or more of mannitol, sorbierite, glucose, lactose, galactolipin, palmitinic acid.
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| CN101406706A (en) * | 2008-11-28 | 2009-04-15 | 哈尔滨工业大学 | Microcapsular ultrasound contrast agent based on surfactant and preparation method thereof |
| CN101596322A (en) * | 2007-06-06 | 2009-12-09 | 南方医院 | Aerial emulsion type ultraphonic contrast agent microballoon and preparation method thereof |
| CN101613365A (en) * | 2009-07-17 | 2009-12-30 | 哈尔滨工业大学 | Based on compound lipid of tetramethylolmethane and preparation method thereof |
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| CN101406706A (en) * | 2008-11-28 | 2009-04-15 | 哈尔滨工业大学 | Microcapsular ultrasound contrast agent based on surfactant and preparation method thereof |
| CN101613365A (en) * | 2009-07-17 | 2009-12-30 | 哈尔滨工业大学 | Based on compound lipid of tetramethylolmethane and preparation method thereof |
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