CN100496615C - Method of preparing ultrasonic contrast agent using mechanical oscillation - Google Patents

Abstract

The present invention relates to a kind of method that utilizes mechanical oscillation to prepare ultrasonic contrast agent, and this method can be used for preparing various ultrasonic contrast agents, comprise the ultrasonic contrast agent (lipid, albumin class, multimer class, surface Active agents, etc.), targeted ultrasound contrast agents, nanoscale microbubble ultrasound contrast agents, and therapeutic ultrasound contrast agents that are also drug or gene targeting carriers; agent method.

Description

A kind of method of utilizing mechanical oscillation to prepare acoustic contrast agent

One, technical field

The present invention relates to acoustic contrast agent, be particularly related to a kind of method of utilizing mechanical oscillation to prepare acoustic contrast agent, this method is applicable to the multiple acoustic contrast agent of preparation, comprises the conventional therapeutic type acoustic contrast agent that strengthens the acoustic contrast agent that tissue develops (lipid, albumin class, polymer class, surfactant-based etc.), targeted ultrasound contrast agent, nanoscale microcapsular ultrasound contrast agent and double medicine or gene targeting vector; The present invention also is specifically related to a kind of method of utilizing mechanical oscillation to prepare the lipid acoustic contrast agent.

Two, background technology

Conventional acoustic contrast agent is a kind of suspension that contains the high concentration microbubble, about 2～4 microns of the average diameter of microbubble, and less than erythrocyte, can be freely by pulmonary circulation, but through blood vessel, be a kind of blood flow tracer.Contrast agent is after peripheral vein injects, because the microvesicle acoustic characteristic different with surrounding tissue, thereby strengthen the echo contrast of blood flow and surrounding tissue, obviously improve ultrasonic image to the dabbling evaluation of tissue blood flow, significantly improve the sensitivity and the specificity of ultrasonic diagnosis, further expand the ultrasonic diagnosis field.

In recent years, along with going deep into of research, it is found that acoustic contrast agent not only can be used for conventional radiography to be strengthened, also can be used for targeting diagnosis and interventional therapy:, the sensitivity of ultrasonic diagnosis and specificity are further improved by preparing targeted ultrasound contrast agent at contrast agent surface combination ligands specific; And the research of interventional therapy constantly enlarges the range of application of acoustic contrast agent, and using value constantly promotes.As: utilize contrast agent microbubble to reduce the ultrasonic cavitation threshold value, thereby promote the therapeutic effect of ultrasound thrombolysis or raising high-intensity focused ultrasound; Or utilize ultrasound wave to destroy the contrast agent microbubble of carrying gene or medicine, and make the release of gene or drug targeting and promote it to penetrate, shift or the drug targeting treatment with mediated gene; Or utilize radiography guiding radio frequency or micro-wave therapeutic and estimate therapeutic effect immediately, etc.

For this reason, external medical institutions prediction acoustic contrast agent will become the common agents in ultrasonic diagnosis or the treatment soon, have huge social benefit and economic worth.

The progress from air micro-bubbles to fluorine carbon microvesicle has been experienced in the development of acoustic contrast agent: the first generation contains the air micro-bubbles contrast agent, as Levovist, Albunex etc.,, persistent period weak point faint because of reinforced effects, diagnostic value is little, withdraws from contrast agent market and research field after 2000 gradually; The fluorine-containing carbon microbubble contrast agent of the second filial generation, not only reinforced effects is remarkable, and the persistent period obviously prolong, for multiple disease clear diagnostic value is arranged.

Another progress of acoustic contrast agent development is the improvement of filmogen.From the history of contrast agent development, the human albumin is as filmogen for the many employings of the nineties initial stage in last century, and the later stage begins to develop to matrix material or polymer material direction.Reason is, considers from secure context, and there is the probability of blood propagation diseases such as propagating acquired immune deficiency syndrome (AIDS), hepatitis B in the human albumin; From the radiography effect, the lipid contrast agent is more stable, more flexible by the film that the macromole lipid constitutes, the radiography better effects if, and the persistent period is longer, more can satisfy clinical needs.Therefore, the contrast agent of new approval listing all belongs to the lipid contrast agent as the SonoVue in the Definity of the U.S. and Imageat, Europe.

At present, existing multiple abroad new generation product goes on the market, and comprises Optison, Definity and the Imageat of the U.S., the SonoVue in Europe; And the domestic not contrast agent of new generation of autonomous listing, the SonoVue that European Bracco company produces is uniquely ratified commercial acoustic contrast agent by China's Ministry of Public Health, and price is very expensive.In addition, contrast agent in the market is not very good on performance yet, and outstanding behaviours is at radiography on the persistent period.It is reported that SonoVue is to only 4～8 minutes effective enhancing time of parenchymatous organ, this is difficult to satisfy the needs of a detailed ultrasonic examination of internal organs (generally needs are 10～15 minutes).From the angle of development, acoustic contrast agent will just more need to improve the stability of contrast agent in blood circulation further in the development of interventional therapy field, prolongs at the body circulation time.

Based on this, we have developed a kind of long-acting lipid acoustic contrast agent " the fat fluorine shows " (Chinese invention patent: patent No. ZL 02 1 33720.9), this contrast agent employing sound method of shaking prepares, have that microvesicle output capacity height, using dosage are little, characteristics such as radiography effect excellence, longer duration, safety are good, cheap, especially it is remarkable to strengthen aspect the time effect at radiography, persistent period in internal organs such as the chambers of the heart, liver, kidney reached more than 30 minutes, was better than domestic and international multiple contrast agent.But in further development process, we find that the sound method of shaking has certain limitation, and outstanding behaviours exists: the 1. technological parameter of sonde-type sound Vibration Meter, comprise power, probe in the position of liquid level, the degree of depth etc., wayward, the technology repeatability is under some influence; 2. sound shakes and is difficult to accomplish the sterile working in the process, and has possibility of metallic pollution, has increased certain difficulty to quality control, the preparation technology of contrast agent; 3. sound shakes and produces more heat in the process, and to the activity of lipid, especially when the contrast agent of some part, medicine or gene is carried in preparation, the activity of part, medicine or the gene that sound is simultaneously shaken produces very big influence.In addition, the sound method of shaking is present most widely used a kind of preparation method, the acoustic contrast agent of the disclosed several types of Chinese invention patent, comprise albumin class (CN1156626, CN1243753), lipid (CN1631444), polymer class (CN1398640), surfactant-based (CN1369311) etc., its preparation process all is the employing sound method of shaking, and shake limitation ubiquity in its preparation process of method of sound.Therefore, it is necessary, also very valuable with the replacement sound method of shaking to seek a kind of better preparation method.

Three, summary of the invention

One of purpose of the present invention is: provide a kind of mechanical oscillation that adopts to replace the method that sonic oscillation prepares acoustic contrast agent.

Two of purpose of the present invention is: adopting a kind of new method for preparing long-acting lipid acoustic contrast agent of mechanical oscillation.

1. adopt mechanical oscillation to prepare the method for acoustic contrast agent

The analysis sound legal system of shaking is equipped with the principle of acoustic contrast agent: the preparation of all kinds acoustic contrast agent all is the positive and negative acoustic pressure of the high frequency conversion that produces when utilizing supersonic oscillations, negative acoustic pressure wherein makes and is present in the gas expansion that contrast agent prepares in the liquid and forms micro-bubble, at this moment, lipid in the preparation liquid or albumin, surfactant, polymer etc. are taken advantage of the occasion to wrap up micro-bubble and are formed stable contrast agent microbubble.In like manner, during the high frequency mechanical oscillation since in the preparation liquid each point stressed the time differently mutually produce different positive/negative pressures, negative pressure wherein also can make the gas that is present in the preparation liquid form micro-bubble.

According to the mechanical oscillation ratio juris, frequency is high more, and the conversion of positive/negative pressure is fast more, and the time of negative pressure is short more, and gas expansion is more little, and the bubble of formation is just more little; Amplitude is more little, and the negative pressure of generation is more little, and under the identical time situation, the gas expansion that is produced by negative pressure is more little, and the bubble of formation is just more little.Because the preparation acoustic contrast agent need form less microbubble, therefore, the mechanical oscillation device of preparation acoustic contrast agent needs higher frequency, lower amplitude.In addition, the mechanical oscillation mode also is a significant effects factor.The mechanical oscillation mode generally is divided into two kinds: reciprocating type and Scroll-tupe, the wherein reciprocating type level that is divided into again is back and forth with vertical reciprocal.In the preparation process of acoustic contrast agent, the eddy current that Scroll-tupe produces causes established contrast agent microbubble to collide mutually and merge, break easily, and therefore reciprocating type vibration is a better choice.

Comprehensive above requirement, we find that capsule-type argental mercury dispenser is to meet the mechanical oscillation device that is used to prepare acoustic contrast agent most in the existing instrument.Capsule-type argental mercury dispenser is that department of stomatology treatment dental caries uses the argental mercury capsule to make it to finish amalgamating special equipment; it adopts reciprocating type mode of oscillation; have frequency of oscillation height, stable performance, protection automatically, characteristics such as cheap, easy to use; its structure mainly comprises motor, chuck (or bayonet socket), protective cover, control panel etc.; only need during use the argental mercury capsule is contained on the chuck; build protective cover, selected duration of oscillation is pressed starting switch and is got final product.Mechanical oscillation device of the present invention converts on the basis of capsule-type argental mercury dispenser just, repacking mainly comprises: what of contrast agent dosage are (1) basis once prepare, select different vibration bottle (the conventional cillin bottle of using), the size of corresponding adjustment instrument comprises the size of motor, chuck and protective cover; (2) according to the effect of contrast agent preparation, before the instrument typing, explore the influence of different operating parameter (comprising frequency of oscillation, amplitude, time) repeatedly, to select optimal working parameter or parameter area to the contrast agent preparation.According to the result who gropes repeatedly, puts into practice, we think that the mechanical oscillation device of preparation acoustic contrast agent should meet the following conditions: (1) mode of oscillation is reciprocating type; (2) frequency of oscillation 〉=3000 time/minute; (3) Oscillation Amplitude: 10～30mm; Its basic preparation method is: the preparation liquid of contrast agent is placed in the vibration bottle, and gas displacement (promptly with required gas will vibrate the air displacement of bottle head) is mechanical oscillation 30～180 seconds afterwards; Or the preparation liquid of contrast agent made freeze dried powder, before the preparation again hydration form preparation liquid, mechanical oscillation is 30～180 seconds then, wherein, gas displacement can be when making freeze dried powder, also can be after hydration forms preparation liquid again.

Mechanical oscillation device and method of the present invention successfully realized one of purpose of the present invention, and it has not only overcome the shake limitation of method of sound previously, makes operation simpler, and performance is more stable, and the technology repeatability is better, and range of application is wider.

According to the principle of existing acoustic contrast agent preparation, mechanical oscillation device of the present invention can be used for preparing present various types of acoustic contrast agent.In the conventional acoustic contrast agent, lipid, surfactant-based, polymer class etc. can directly be utilized this device to add common vibration bottle to prepare; The albumin class owing to need higher temperature to make the albumin degeneration, therefore need increase frictional heat by add small bead in the vibration bottle in preparation process.The preparation of targeted ultrasound contrast agent has two kinds of selections, and a kind of is at present the most frequently used: add ligands specific after conventional acoustic contrast agent preparation is finished, by preparing behind this device vibration certain hour; Another kind is to add ligands specific in advance, ligands specific in the combination when forming contrast agent microbubble in the preparation liquid before contrast agent preparation is finished.A kind of selection in back be adopted in the past sound shake legal system be equipped with institute can not, its advantage: the one, reduced the preparation link, the 2nd, reduced the destruction for preparing microvesicle in the link.

This mechanical oscillation device biggest advantage is that heat production is few in the contrast agent preparation process, therefore especially is suitable for preparing the acoustic contrast agent of double medicine or gene targeting vector.The mode that contrast agent microbubble is carried gene or medicine is divided into two kinds: stick method and integration method, wherein integration method has remarkable advantages, reason is: stick method and only by simple mixing medicine or gene are attached on the contrast agent microbubble surface, binding capacity is few on the one hand, adherent on the other hand medicine or gene combine insecure with microvesicle, after intravenous injection, under impacting, blood flow is easy to come off, and targeting is poor; And integration method both can be attached on the microvesicle surface with medicine or gene, more it can be incorporated on the microvesicle film or be wrapped in the microvesicle, when binding capacity is increased, make targeting better again.But when previously employing sound shakes method,, therefore can not add medicine or gene simultaneously in the preparation, can only adopt and stick the acoustic contrast agent that method prepares hold concurrently medicine or gene targeting vector owing in preparation process, produce higher temperature.And mechanical oscillation device of the present invention quantity of heat production in the contrast agent preparation process is few, therefore can adopt the acoustic contrast agent of double medicine of integration method preparation or gene targeting vector.

2. new method for preparing long-acting lipid acoustic contrast agent

For verifying that mechanical oscillation device of the present invention prepares the effect of acoustic contrast agent, also in order to optimize the preparation technology of " the fat fluorine shows ", two of purpose of the present invention is: replace the sonic oscillation instrument with mechanical oscillation device of the present invention, the preparation technology of corresponding adjustment " the fat fluorine shows " explores a kind of new method for preparing long-acting lipid acoustic contrast agent.

After the adjustment, " the fat fluorine shows " key step of preparation is:

(1) hydration:, form the lipid suspension with the lipid in the contrast agent filmogen, high molecular polymer and aqueous solution or the abundant mixing of nonaqueous solvent;

(2) lyophilization: the lipid suspension lyophilization of step (1) is formed lyophilized powder;

(3) rehydrated: that the lyophilized powder of step (2) with the abundant mixing of aqua liquid, is formed the lipid suspension again;

(4) gas displacement: the air that will fill head in the vibration bottle of lipid suspension of step (3) is replaced with fluoro-gas;

(5) mechanical oscillation: the vibration bottle of step (4) is placed on the chuck of mechanical oscillation device, carry out mechanical oscillation, promptly form the acoustic contrast agent that contains a large amount of microbubbles after the vibration by the running parameter that sets.

Lipid described in the above-mentioned steps (1) is phospholipid and/or additives.Phospholipid has parents' molecular characterization, biological degradability and biocompatibility, in the preparation process of acoustic contrast agent, form the film of unimolecule or bilayer structure, parcel gas forms microbubble, thereby be necessary composition in the preparation of lipid acoustic contrast agent, wherein, the most frequently used phospholipid is lecithin (being phosphatidylcholine), comprise natural, as Ovum Gallus domesticus Flavus lecithin, soybean lecithin; Semisynthetic, as part or abundant hydrogenant lecithin; And synthetic phospholipid, as dipalmitoyl phosphatidyl choline (DPPC), distearoyl phosphatidylcholine (DSPC).Other phospholipid commonly used also comprises phosphatidic acid, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine, phosphatidyl glycerol, phosphatidylinositols, cuorin, sphingomyelins, and the fluorinated analogues of lecithin and any above-mentioned phospholipid.Carry the targeted ultrasound contrast agent of ligands specific for preparation, phospholipid can also carry polymeric chain, as carries Polyethylene Glycol (PEG), polyvidon (PVP) and the derivant thereof with amphipathic characteristic.The principle of repelling each other according to the same sex, increase the stability of acoustic contrast agent, it is very favorable adding in the composition of lipid or all utilizing the phospholipid of band net charge (as negative charge), as naturally occurring (deutero-as Semen sojae atricolor or egg yolk), semisynthetic (as part or fully hydrogenant) and synthetic Phosphatidylserine, phosphatidyl glycerol, phosphatidylinositols, phosphatidic acid and cuorin.Additives itself do not form unimolecule or bilayer structure, thereby it is necessary to be not that contrast agent prepares, but it can participate in the immobilized artificial membrane, regulates the function of film, can regulate flowability, the permeability of film as cholesterol, 18-amine. can change (containing positive charge) charge property on film surface.Lipid of the present invention comprises a kind of phospholipid at least, is preferably synthetic phospholipid; If single phospholipid, then preferably with the phospholipid of net charge; If the mixed phosphatide that a plurality of phospholipid (two or more) form, then at least a is the phospholipid of band net charge.

High molecular polymer described in the above-mentioned steps (1) is a Polyethylene Glycol, and preferred scheme is Macrogol 4000 (PEG4000).Using to the contrast agent film of high molecular polymer provides a supporting structure, simultaneously, because bigger sterically hindered of the formed hydrophilic barrier of the hydrophilic Polyethylene Glycol film of one deck and peg molecule wrapped up on the film surface, can prevent that contrast agent is by the identification of plasma protein and reticuloendothelial system and engulf, the holdup time of contrast agent in blood circulation prolonged, in reticuloendothelial systems such as liver, spleen, bone marrow, engulf minimizing, increase the body internal stability of contrast agent, prolong the effective enhancing time of contrast agent in the tissue development.

Aqueous solution described in the above-mentioned steps (1) is selected from water (comprising water for injection, deionized water, distilled water, ultra-pure water etc.), normal saline, the mixture (wherein the shared volume of glycerol is 5%～20%) of saline/glycerol and the mixture (volume ratio is 8:1:1) of saline/glycerin/propylene glycol; Described nonaqueous solvent is selected from propylene glycol, Liquid Macrogol, the tert-butyl alcohol, the positive tert-butyl alcohol.In a more preferred embodiment, described aqueous solution is selected water.

Lipid, high molecular polymer and aqueous solution described in the above-mentioned steps (1) or the blended ratio of nonaqueous solvent are to contain lipid 1～10mg, pbz polymer polymer 50～200mg in every ml water solution or the nonaqueous solvent.

Abundant mixing described in the above-mentioned steps (1) comprises the various means that those skilled in the art grasp, and wherein the most frequently used is heating and vibration, and heating-up temperature should be a little more than the highest phase transition temperature of selected lipid.

Lyophilization described in the above-mentioned steps (2) is a routine techniques, cryodesiccated purpose: the one, and the dissolubility of increase lipid, easier dispersion when making lipid rehydrated, formed lipid suspension is more tiny, even; The 2nd, the surface area of increase lipid.After the lyophilizing, lipid is more tiny, but relative surface area is bigger, therefore, can form more, littler microvesicle when the preparation contrast agent, and the productive rate of contrast agent microbubble and quality are obviously improved.

Aqua liquid described in the above-mentioned steps (3) is selected from the mixture (volume ratio is 8:1:1) of hyperosmotic glucose, hyperosmotic glucose/glycerin/propylene glycol and the mixture of above-mentioned solution and foaming agent.Wherein, hyperosmotic glucose is a concentration〉10% glucose, fructose and isomer thereof, polysaccharide; Foaming agent is non-ionic surfactant, comprises tween ₈₀, span ₈₀Deng, select tween when specifically using ₈₀, the ratio of adding is to contain tween in every milliliter of aqua liquid ₈₀0.001～0.01ml.In a more preferred embodiment, described aqua liquid is the mixture (volume ratio is 8:1:1) of hyperosmotic glucose/glycerin/propylene glycol, and wherein the concentration of hyperosmotic glucose is 20%～50%.The amount that adds aqua liquid contains lipid 1～3mg for adding in the every milliliter of lipid suspension in back, and preferred scheme is 1.5mg.The purpose of using hyperosmotic glucose is in order to increase the viscosity of solution, to reduce the mutual fusion tendency of contrast agent microbubble, simultaneously the hydrogen bond on the sugar also directly and the interaction of hydrogen bond of lipid, both have strengthened the stability of contrast agent microbubble jointly.The adding of glycerol and propylene glycol, the one, increased the viscosity of solution, reduce the mutual fusion tendency of contrast agent microbubble; The 2nd, can be used as co-emulsifier, make the easy film forming of lipid, to increase the productive rate of microvesicle.Use the purpose of foaming agent: the one, in order to increase the output capacity of microvesicle; The 2nd, adipose membrane is played Stabilization, to prolong the time of contrast agent in the tissue development.

Abundant mixing described in the above-mentioned steps (3) comprises the various means that those skilled in the art grasp, and wherein the most frequently used is heating and vibration.Because lipid easier dispersion after lyophilization, therefore, the abundant mixing difficulty of this step is little, and get final product generally only hand a moment.

Vibration bottle described in the above-mentioned steps (4) is conventional uses cillin bottle, and its size should be fit to the chuck of the mechanical oscillation device that adopts.In addition, experiment finds, the volume of vibration bottle inner lipid suspension accounts for the volumetrical ratio of whole vibration bottle has very big influence to the preparation effect of contrast agent.The vibration bottle ratio that the inner lipid suspension is shared is more little, and the amplitude of lipid suspension motion is with regard to big more (it is big more to be equivalent to amplitude) during mechanical oscillation, and formed microbubble is just big more; On the contrary, the shared ratio of lipid suspension is big more, and formed microbubble is just more little.Therefore, the volume of vibration bottle inner lipid suspension account for a whole vibration bottle volumetrical ratio should be suitable.Through repeatedly practising, this ratio should be preferably in about 40% within 30%～70% scope.

Fluoro-gas described in the above-mentioned steps (4) comprises pfc gas, sulfur fluoride gas, and preferred scheme is perfluoropropane gas (C ₃F ₈).Fluoro-gas is a noble gas, and molecular weight is big, and dissolubility and dispersivity in blood are poor, good stability.The method of gas displacement has a variety of, the method that laboratory is commonly used has: the bottle that 1. will vibrate is upright, directly in the vibration bottle, inject a certain amount of fluoro-gas (volume is suitable with the air of vibration bottle head), air displacement is come out by action of gravity (fluoro-gas is heavier than air manyly); 2. utilize tee T, extract the air in the vibration bottle earlier out, the fluoro-gas of the isodose that reinjects; The industry preferable methods is that the vibration bottle of packing is put into lyophilization is indoor, and the air with fluoro-gas displacement vibration bottle head also can use other method well known by persons skilled in the art.

Running parameter described in the above-mentioned steps (5) comprises frequency, amplitude, the time of mechanical oscillation, and its medium frequency is 3000～8000 times/minute, and amplitude is 10～30mm, and the time is 30～90s.

Need to prove, more than only be key step among the contrast agent preparation technology, other also comprises:

(1) asepticize of lipid suspension is handled: the method that often adopts high-temperature pressurizing filtration sterilization before packing; Also can adopt the method for end sterilization eventually, the vibration bottle that is about to seal after the packing is sterilized in autoclave sterilizer, because autoclaving can cause lipid degraded to a certain degree, so the GPRS condition of sterilizing well, generally be 126～130 ℃ of sterilization 1～10min; Or use above-mentioned two kinds of methods simultaneously.

(2) packing of lipid suspension and sealing: according to the difference of preparation technology's flow process, the packing of lipid suspension can be in the key step (1) of above-mentioned preparation afterwards, also can be in the key step (3) of above-mentioned preparation afterwards.The former advantage is: only need the primary freeze drying process just can finish hydration → lyophilization → gas displacement → overall processes such as sealing, just can be used as the lyophilized powder manufactured goods behind the autoclaving; Shortcoming is: these lyophilized powder manufactured goods aqua liquid of need providing and delivering, want during use earlier aqua liquid is added in the lyophilized powder finished product, and shaking up the back mechanical oscillation just can become needed contrast agent finished product in tens of seconds.The latter's shortcoming is: the technological process relative complex, to successively pass through hydration → lyophilization → rehydrated → packing → freeze drying chamber and carry out gas displacement → sealing → processes such as asepticize processing, just can become manufactured goods, and manufactured goods are water preparation, the shelf-life is not as freeze dried powder; Advantage is: packing and use are simpler, the aqua liquid of need not providing and delivering, and the direct mechanical vibration got final product in tens of seconds during use.

Characteristics such as the present invention adopts that mechanical oscillation prepares that long-acting lipid acoustic contrast agent " fat fluorine show " has kept not only that former method microvesicle output capacity height, using dosage are little, radiography effect excellence, longer duration, safety are good, cheap also have following characteristics:

(1) adopts the mechanical oscillation device to replace the sonic oscillation instrument, successfully overcome the limitation of legal system when being equipped with of shaking of sound previously;

(2) contrast agent microbubble of this method preparation output capacity height not only, and the particle size range of microvesicle is narrow, size evenly, the preparation back can be used at once; And the sound legal system of shaking is equipped with the back owing to there is the big microvesicle of some to exist, will be by leaving standstill or filterable way could be used after removing big microvesicle before using;

(3) this method can add ligands specific in containing the vibration bottle of lipid suspension, the long-acting targeting lipid of disposable preparation acoustic contrast agent; Also can in containing the vibration bottle of lipid suspension, add required medicine or gene, with medicine or gene integration on film or be wrapped in the film, the hold concurrently long-acting lipid acoustic contrast agent of medicine or gene targeting vector of disposable preparation;

(4) because the mechanical oscillation device cheap (less than 1000 yuan) of miniaturization, therefore, " the fat fluorine shows " of the present invention's preparation can make the freeze dried powder or the aqueous injection of lipid suspension earlier, before the use again mechanical oscillation be prepared into contrast agent.Compare with the freeze dried powder of the past contrast agent microbubble, preparation technology is simpler for this method, and cost of manufacture is lower, but is convenient to storage and transport equally.

Four, description of drawings

Fig. 1 is a kind of mechanical oscillation device of the present invention;

Fig. 2 is for adopting the image (* 400) of lipid acoustic contrast agent microvesicle under light microscopic of mechanical oscillation instrument preparation;

Fig. 3 is contrast agent strengthens video picture to rabbit liver essence a dynamic image.

Referring to accompanying drawing 2, show among the figure after contrast preparation dilutes 100 times and under 400 times of light microscopics, observe the microvesicle branch Cloth is even.

Referring to accompanying drawing 3, this figure is zoopery radiography effect figure, and this accompanying drawing shows that contrast preparation can increase for a long time Strong hepatic parenchymal GTG video picture is dynamically observed, 60min behind the radiography, and still visible contrast preparation is little in the inferior caval vein The echo of bubble (A, B, C, D, E, F be respectively radiography before, 11s, 1min behind the radiography, 12min, 30min, 60min).

Five, the specific embodiment

Take by weighing two palmityl phosphatidyl glycerol (DPPG) 30mg, DSPC 30mg, PEG40001940mg with the 100ml conical flask, add 40ml deionized water after the preheating in 60 ℃ of waters bath with thermostatic control, conical flask is put into 60 ℃ of water bath with thermostatic control shaking tables, regulate the about 200rpm of speed of shaking table, vibration 10～20min disperses fully until lipid.With pipettor the lipid suspension branch in the conical flask is packed into (the about 3.5ml of this cillin bottle actual volume is suitable as the vibration bottle) in 40 bottles of 1ml cillin bottles, lyophilization becomes freeze dried powder.The mixture (volume ratio is 8:1:1) that adopts hyperosmotic glucose/glycerin/propylene glycol is as aqua liquid, and wherein hyperosmotic glucose is 20% glucose, adds aqua liquid by every bottle of 1.2ml, with the light mixing of have gentle hands.Cillin bottle is upright, directly in the vibration bottle, inject about 2.5ml perfluoropropane, by action of gravity air displacement is come out.(annotate: the frequency of oscillation of this mechanical oscillation device and amplitude are fixing after the instrument molding with the mechanical oscillation device with cillin bottle, be respectively frequency 4100 times/minute, the chuck of amplitude 15 ± 1mm) fixes, the time of setting mechanical oscillation is 45s, opening power, starting switch is finished oscillatory process by the default time, promptly prepares required lipid acoustic contrast agent.

After the contrast agent preparation, with om observation microvesicle size, form, blood counting chamber is measured microbubble concentration, laser particle size analyzer mensuration particle diameter and distribution, surface potential, pH value; As laboratory animal, carry out ultrasonic contrast by the dosage of 0.01ml/kg with new zealand white rabbit, observe the reinforced effects of this contrast agent the rabbit normal liver through auricular vein.The result: 1. to several μ m, all less than 8 μ m (see figure 2)s, mean concentration is 2.56 ± 0.15 * 10 to the particle diameter of contrast agent microbubble from hundreds of nm ¹⁰/ ml, mean diameter is 1.67 ± 0.11 μ m, surface potential is-37.5mV ± 0.3mV that pH value is 6.46 ± 0.21; 2. this contrast agent can strengthen hepatic parenchymal GTG video picture for a long time, the enhancing persistent period reaches the above (see figure 3) of 60min, and safety is good, show as: whole angiographic procedure, all animal lives are levied steadily, the sick inspection in experiment back, and institute is in a organized way, comprise the heart, liver, kidney, lung, reach micro-pathologic finding substantially and there is no unusually; 3. no matter technology favorable reproducibility after the contrast agent preparation, is physical property, or radiography effect basically identical all.

Claims (8)

1. A method for preparing an ultrasound contrast agent by mechanical oscillation, characterized in that it comprises the following steps: (1) Hydration: Fully mix the lipids and polymers in the film-forming material of the ultrasound contrast agent with an aqueous solution or a non-aqueous solvent to form a lipid suspension; the lipids are phospholipids or phospholipids and additives The mixture; the high molecular polymer is polyethylene glycol; the aqueous solution is selected from water for injection, deionized water, distilled water, a mixture of saline/glycerin and a mixture of saline/glycerol/propylene glycol, a mixture of saline/glycerol The volume of glycerol in the mixture is 5% to 20%, the volume ratio of saline/glycerin/propylene glycol in the mixture of saline/glycerin/propylene glycol is 8:1:1, and the non-aqueous solvent is selected from propylene glycol, polyethylene glycol 300, tert-butanol, n-tert-butanol; (2) freeze-drying: the lipid suspension of step (1) is freeze-dried to form freeze-dried powder; (3) Rehydration: fully mix the lyophilized powder in step (2) with a hydration solution to re-form a lipid suspension, the hydration solution is selected from a mixture of hypertonic sugar, hypertonic sugar/glycerin/propylene glycol; Among them, hypertonic sugars are glucose with a concentration of >10%, fructose and its isomers with a concentration of >10%, polysaccharides with a concentration of >10%; (4) gas replacement: the air in the head of the shaking bottle filled with the lipid suspension of step (3) is replaced with fluorine-containing gas; (5) Mechanical oscillation: place the oscillation bottle in step (4) on the chuck of the mechanical oscillation device, perform mechanical oscillation according to the set working parameters, the mechanical oscillation mode is reciprocating, and the oscillation frequency is 3000-8000 times/ minutes, the range is 10-30mm, the time is 30-180 seconds, After oscillation, an ultrasound contrast agent containing a large number of tiny air bubbles is formed. 2. The method according to claim 1, wherein the lipids in step (1) are synthetic phospholipids comprising at least one phospholipid with a net charge. 3. The method according to claim 1, wherein the high molecular polymer in step (1) is polyethylene glycol 4000. 4. The method according to claim 1, wherein in the step (1), the lipid, the polymer and the aqueous solution or the non-aqueous solvent are mixed in a ratio of 1 to 10 mg of the lipid in each milliliter of the aqueous solution or the non-aqueous solvent, containing High molecular polymer 50 ~ 200mg. 5. The method according to claim 1, wherein the hydration solution described in step (3) is a mixture of hypertonic sugar/glycerin/propylene glycol, wherein the concentration of hypertonic sugar is 20%-50%; The amount is 1-3 mg of lipid per milliliter of lipid suspension after addition. 6. The method according to claim 1, wherein the oscillating bottle described in the step (4) is conventionally used in vials, its size should be suitable for the chuck of the mechanical oscillating device adopted, and the volume of adding the lipid suspension accounts for The proportion of the volume of the entire shaking bottle should be within the range of 30% to 70%. 7. The method according to claim 1, wherein the fluorine-containing gas in step (4) is perfluorocarbon gas or sulfur fluoride gas. 8. The method according to claim 1, wherein the fluorine-containing gas in step (4) is perfluoropropane gas.

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