CN1285383C

CN1285383C - Method and use of closed hydrophilic and lipophilic liquid-phase hollow capsules with cores

Info

Publication number: CN1285383C
Application number: CN 200410060609
Authority: CN
Inventors: 周翔
Original assignee: Individual
Current assignee: Individual
Priority date: 2004-07-22
Filing date: 2004-07-22
Publication date: 2006-11-22
Anticipated expiration: 2024-07-22
Also published as: CN1613504A

Abstract

The present invention relates to a method for forming vesicle bubbles by cavitating the self-sealing small body liquid phase cores of hydrophilic and fat-philic molecules. Film-forming materials are dissolved in chloroform and/or methanol, and are sprayed, dried and formed into a film on tiny biodegradable water-soluble powder granule carriers at the phase transition temperature from a gel state to a liquid crystal state in the state of pumping vacuum; tiny water-soluble powder granules are loaded in a container, is placed in vacuum and is sealed; gas and water media of a certain volume are orderly injected at the temperature lower than the phase transition temperature from the gel state to the liquid crystal state; the container is oscillated manually until the tiny water-soluble powder granules are dissolved; the liquid phase core cavitated self-sealing small body vesicle bubbles of hydrophilic and fat-philic molecules are formed. The present invention has the advantages of low cost, safety, no toxicity, easy preparation and subpackage because of the dry powder preparation type, and best use effect, and is used for transporting oxygen in veins when used for clinical treatment.

Description

The self-enclosed corpusculum liquid phase of hydrophilic lipophilic core cavitation becomes the method and the application of vesicle

Technical field

The present invention relates to a kind of method that makes the self-enclosed corpusculum liquid phase of hydrophilic lipophilic amphiphatic molecule core cavitation form the capsule bubble.The small capsule bubble of the present invention is applied in and is used for ultrasonic contrast imaging on the medical diagnosis, is used for vein oxygen therapy treatment anoxia disease on clinical treatment.

Background technology

The microbubble (capsule bubble) of small capsule bubble or title shell parcel has a wide range of applications at medical field.Found in recent years can significantly improve the ultra sonic imaging comparative through the microbubble of intravenous injection microbubble or shell parcel.Ultrasonic contrast is to make ultrasonic reflection or the enhanced a kind of technology of scattering with acoustic contrast agent, gas is compared with tissue with surrounding liquid in the microvesicle that contrast agent contained or that produce that uses in the ultrasonic contrast, in ultrasonic sound is raised, compare and have exponential compressibility difference with surrounding tissue, blood constituent, produce intensive reflecting interface, microvesicle is graininess echo to be strengthened or cloud echo, the echo potentiation of contrast agent can more clearly illustrate the chambers of the heart or endovascular blood, the principle of Here it is ultrasonic contrast imaging.

Early stage acoustic contrast agent micro-bubble diameter is all greater than 10 microns, can not be by the blood capillary of pulmonary circulation, be the dextrocardiography agent, and be free bubble, as the hydrogen peroxide ultrasonic contrast, be through the intravenous injection hydrogen peroxide, in the effect of erythrocyte hydrogen peroxide enzyme, decomposition discharges oxygen, and wherein part combines with hemoglobin, all the other are the free state disperse in blood, cause ultrasonic boundary reflection; As the carbon dioxide ultrasonic contrast, be that sodium bicarbonate solution mixes with various acid solutions (acetic acid, hydrochloric acid, vitamin C etc.), carbon dioxide gas forms ultrasonic boundary reflection after intravenous injection.

Because concentrating in logic naturally, the unstability of free bubble and the restriction of bubble diameter, researcher attention can spontaneous sealing form on the hydrophilic lipophilic amphiphatic molecule of miniature spherical liquid phase corpusculum.At present, can be in liquid phase environment, can be called the self-enclosed corpusculum of hydrophilic lipophilic amphiphatic molecule generally by spontaneous sealing formation microsphere sample corpusculum, mainly contain two classes: a class is called liposome (Liposome), another kind of liquid phase corpusculum (the Non-ionic Surfactant based Vesicles that is called based on non-ionic surface active agent, Niosome, referring to document: UchegbuIF, Vyas S Non-ionic surfactant based vesicles (niosomes) in drugdelivery, International Journal of Pharmaceutics 172 (1998) 33-70) abbreviate the non-ionic surface active corpusculum as.Both mix the non-ionic surface active phospholipid mixing corpusculum of formation when preparing in addition in addition.In fact the amphiphatic molecule that forms this self-enclosed corpusculum of hydrophilic lipophilic amphiphatic molecule all belongs to the surfactant category.A large amount of surfactants, comprise a lot of non-ionic surface active agent molecules, various types of lipid molecular particularly phospholipid be hydrophilic lipophilic both sexes bilayer spline structure.These surfactant molecules, after adding certain film additive, its hydrophilic lipophilic amphiphatic molecule imports certain mechanical energy and stirs in aqueous medium, can spontaneous sealing form the hydrophilic lipophilic bilayer microsphere spline structure with liquid phase core.These physicochemical properties have determined them to have the application prospect of most suitable film parcel gas.The key of problem is how to make its liquid phase core cavitation and form small capsule bubble.

The non-ionic surface active corpusculum is selected the seventies and find for the researcher of the industry of making up most.This class non-ionic surface active corpusculum and liposome are similar,, are imported certain mechanical energy and stir in aqueous medium by the hydrophilic lipophilic amphiphatic molecule of non-ionic surface active agent, can spontaneous sealing form bilayer microsphere spline structure.Because the formation and the hydrophilic-lipophilic balance value (HLB, hydrophilic-lipophilicbalance value) of non-ionic surface active corpusculum have substantial connection, it is generally acknowledged that the HLB value is suitable for the formation of non-ionic surface active corpusculum between 4-10.Therefore for the surfactant of high HLB value, often the other types surfactant mixing preparation with low HLB value goes out appropriate H LB value, as lecithin being added non-ionic surface active agent allotment appropriate H LB, should be phospholipid-non-ionic surface active mixing corpusculum in this case on the corpusculum stricti jurise of Xing Chenging.Equally, when the preparation liposome, often also need to add some nonionic surfactant as film modified.This also can think aforesaid mixing corpusculum.The spontaneous sealing corpusculum of hydrophilic lipophilic amphiphatic molecule (non-ionic surface active corpusculum and liposome) preparation method is all similar, shakes as rotary evaporation, sound and handles etc.The conventional non-ionic surface active corpusculum and the preparation of liposome are carried out in two steps suddenly: 1) film forming: the non-ionic surface active agent filmogen and/or the liposome filmogen of deployed HLB value are dissolved in the organic solution, and place in the Rotary Evaporators, rotary evaporation, film forming is in container bottom; 2) adult: inject aqueous medium in the container of tunicle, mechanical agitation formed the liquid phase core corpusculum with hydrophilic lipophilic bilayer more than 1 hour.What should emphasize is no matter non-ionic surface active corpusculum or liposome, and no matter film forming procedure still becomes the corpusculum process, all is to carry out on the liquid crystal state phase transition temperature at gel state, could form the little body structure of liquid phase core like this.Conceptive from the science strictness, phospholipid-non-ionic surface active mixing corpusculum that the liposome (Liposome) of general meaning, non-ionic surface active corpusculum (Niosome) or both mix formation is the liquid phase notion, is liquid phase environment inside and outside its hydrophilic lipophilic layers of amphiphilic molecules.The diameter of this class corpusculum can reach the 3-5 micrometer range approximately.Therefore to make the self-enclosed corpusculum liquid phase of hydrophilic lipophilic amphiphatic molecule core cavitation form small capsule bubble, must adopt and generally prepare liposome (Liposome), non-ionic surface active corpusculum (Niosome) or both mix the phospholipid-non-ionic surface active mixing corpusculum diverse ways of formation.

The method for preparing film parcel microbubble at present both at home and abroad mainly contains three classes: 1) sound shakes: utilization sound Vibration Meter probe directly inserts and contains in the liquid of certain film forming surfactant, the vibration of introducing high strength supersonic, utilize the ultrasonic cavitation effect to reach the purpose of film parcel micro-bubble, as by the Chinese invention patent prospectus, the acoustic contrast agent that application number 02133720.9, application for a patent for invention numbers 96106566.4 and application for a patent for invention number 01103726.1, application for a patent for invention numbers 98105120.0 etc. relates to, all employing sound Vibration Meter carries out the sound cavitation of shaking and forms micro-bubble; 2) lyophilization: as Italian acoustic contrast agent Sonovue and as application for a patent for invention numbers 98105120.0, utilize cryodesiccated method, make phospholipid plastid or the sound albumin microsphere bubble that shakes keep the space structure state of vacuolation; 3) adopt special equipment to carry out the liposome cavitation, as the invention of the related blanketing gas liposome of application for a patent for invention 94192405.X.

These three class methods, the most commonly used with sound Vibration Meter preparation, but ultrasonic probe is under the very high energies vibration, the easy residual grain of residual titanium metal in vibrating liquid, and also preparation process is more loaded down with trivial details, is difficult for convenient preparation and packing.And the small capsule bubble of having prepared, in packing and storing process, the part of inevitably will losing and break, the bubble of preparing at once is not abundant.Therefore only just can produce micron-sized bubble with the energy that manually shakes in use is ideal approach.In addition, domestic application for a patent for invention number 02133720.9 and the used phospholipid filmogen of Sonovue and domestic patent application number 02133720.9 is consistent, involves great expense.Freeze-drying method, though be beneficial to packing, the technological requirement height, and the freeze drying equipment input of producing in batches is also expensive, and as Sonovue, the price of a Sonovue is domestic to be about 730 yuan.The invention of the blanketing gas liposome that application for a patent for invention 94192405.X is related needs purpose-made device and special technology, prepares also more loaded down with trivial detailsly, has high input.

Be metabolic process in vital movement in addition, body need constantly consume oxygen, as go out acute clinically, chronic cardio cerebrovascular affection, paralysis of respiratory muscle, asphyxiating pathological changes, and often cause whole body or local organization oxygen content obviously to reduce, be in anaerobic condition.Anoxia can cause all infringements of body tissue, as cytopathy, necrosis, vascular endothelial injury, thrombosis, systemic inflammatory response etc., even causes body death.As 25 microns of the blood capillary radiuses of people's ectocinerea, average distance is 60 microns between blood capillary, under the normal condition, and 30 microns of effective disperse radiuses of dissolved oxygen, partial pressure of oxygen is 2Kpa, does not have the anoxic zone.If the oxygen supply deficiency takes place, the oxygen diffusing capacity descends, and occurs clinical symptoms when partial pressure of oxygen is reduced to 1.33Kpa, partial pressure of oxygen is reduced to 0.53Kpa generation loss of consciousness when following.Have many people to conceive the venous channel oxygen supply for many years, blood transfusion is exactly a kind of important complementary therapy.Human hydrogen peroxide, ultraviolet radiation haemodialysis anoxia disease were arranged in the seventies in 19th century.The development of artificial blood such as the complex that carries oxygen (porphyrin compound) of fluorocarbons, animal hematoglobin and synthetic etc., material that can dissolution of high-concentration oxygen, but reason is promoted eventually owing to having various shortcoming and involve great expense etc.In recent years have researcher to adopt, the vein oxygen supply device of dissolved oxygen jar or hyperoxia solution is to produce certain density active oxygen by reciprocating type printing opacity blood bag after multispectral effect such as illumination such as grade, but the concentration of dissolved oxygen is limited, during infusion, pressure reduces in the body, and often dissolution rate more descends; Therefore, there is no preferably now, mode realizes vein oxygen therapy.

Summary of the invention

The objective of the invention is to overcome in the above-mentioned background technology weak point of little air bag of preparation or microbubble and a kind of method that makes the self-enclosed corpusculum liquid phase of hydrophilic lipophilic amphiphatic molecule core cavitation form small capsule bubble easily be provided:

Another object of the present invention relates to the application clinically of described small capsule bubble, as is used for ultrasonic contrast and cures by oxygen therapy through vein.

But cheap, the avirulence biological metabolism with the small capsule bubble materials of method for preparing, be easy to preserve and packing, do not have that metal object is polluted, diameter is below 10 microns.

The objective of the invention is to reach by following measure: a kind of method that makes the self-enclosed corpusculum liquid phase of hydrophilic lipophilic core cavitation become vesicle, the self-enclosed corpusculum of described hydrophilic lipophilic amphiphatic molecule is meant by non-ionic surface active agent, cholesterol and/or phospholipid filmogen to be formed;

The method of the small capsule bubble of described formation comprises the steps:

(1) filmogen is dissolved in the organic solutions such as chloroform and methanol, under the evacuation state, at gel state on the liquid crystal state phase transition temperature, utilize the spray drying film forming on the small powdered granule carrier of biodegradable water solublity, described filmogen: the water-solubility carrier mass ratio is 1: 50-500000;

(2) with the small powdered granule of above-mentioned biodegradable water solublity, pack in certain volumetrical container, put vacuum, and seal, be lower than gel state under the liquid crystal state phase transition temperature, successively inject specific gas and aqueous medium, room temperature is manually concussion down, and after water miscible subparticle dissolving, wrap film is subsided, the strong concussion of liquid gas interface makes gas enter the self-enclosed corpusculum of hydrophilic lipophilic amphiphatic molecule, and then forms the self-enclosed corpusculum of hydrophilic lipophilic amphiphatic molecule of liquid phase core cavitation.

The self-enclosed corpusculum of hydrophilic lipophilic amphiphatic molecule in the core of liquid phase described in technique scheme cavitation is meant that non-ionic surface active corpusculum (Niosome) or liposome (Liposome) or both mix the phospholipid non-ionic surface active mixing corpusculum of formation.

At the corpusculum of non-ionic surface active described in the technique scheme (Niosome), used non-ionic surface active agent selects one or more allotments in the following type and forms with cholesterol film surface additive; (1) class of department (Span) serial nonionic surfactant is as class of department 20, class of department 40, class of department 60, class of department 80, class of department 85 etc.; (2) the serial nonionic surfactant of tween (Tween), as polysorbas20, polysorbate40, polysorbate60, Tween 80, sorbimacrogol oleate100, polysorbate85 etc.; (3) Brij series nonionic surfactant, as Brij 30, Brij 40, Brij 60, Brij 76, Brij 78, Brij 93 etc.; (4) other: Cremophor EL.PluronicF68, albumin.

At liposome described in the technique scheme; comprise that following all kinds of phospholipid and other film additives constitute; such as comprising following all kinds of phospholipid or lipoid; such as: lecithin; the phosphatidyl glycol amine; cholesterol; cholesterol acetyl fat; cupreol; natrii tauroglycocholas; Yolk lecithin; phosphatidylinositols; sphingomyelin; sphingo; two whale vinegar phospholipid; myristoyl lecithin; stearmide; oleic acid list phospholipid; vitamin E and other people worker's synthetic phospholipid are as 1.2-palmityl-sn-glyceryl-3-phosphatidic acid glyceryl-sodium salt (DPPGNa); 1.2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine (DSPC); 1.2-two palmityls-sn-glyceryl-3-phosphatidic acid glyceric acid sodium salt (DPPANa); 1.2-palmityl-sn-glyceryl-3-3-phosphatidylcholine (DPPC), or the mixture of above-mentioned part of compounds.

In phospholipid described in the technique scheme-non-ionic surface active mixing corpusculum phospholipid hydrophilic-the hydrophilic lipophilic amphiphatic molecule allotment of lipophilic amphiphatic molecule and non-ionic surface active agent ratio is 1-500: 1.

At biodegradable water solublity powdery molecule carrier described in the technique scheme is following any or several mixture: polylactic acid (PLA), polylactide, polycaprolactone (PCL), polyglycolic acid (PGA), poly hydroxybutyric acid (PHB), poly-hydroxyl valeric acid (PVAC), poly-capric acid (PDA), the copolymer p LGA of polylactic acid and polyglycolic acid, the copolymer of polylactic acid and poly hydroxybutyric acid, Polyisohexylcyanoacrylate (PIHCA), polylactide Acetic acid, hydroxy-, bimol. cyclic ester (PLCG) etc., poly-anhydride, semisolid poe (POE), Polyethylene Glycol is (as cetomacrogol 1000, Macrogol 2000, Macrogol 3000, Macrogol 4000, Polyethylene Glycol 5000, polyethylene glycol 6000, Polyethylene Glycol 8000), polyvidon, dibenzylatiooluene, sorbitol, sodium carboxymethyl cellulose, hetastarch, carboxymethyl starch sodium, alginate; Glucose, galactose, fructose, maltose, sucrose, maltodextrin, glucosan, water-soluble chitosan; Sorbitol; Sodium chloride.

Wrap up the particle size distribution of biodegradable water solublity powdery molecule carrier at micron order and/or nanoscale at biodegradable water solublity powdery molecule carrier and film described in the technique scheme.

At injecting gas described in the technique scheme is a kind of any or several in fluoroalkane hydro carbons gas (as perfluoromethane gas, hexafluoroethane gas, perfluoropropane), air, oxygen, nitrogen, carbon dioxide gas, nitric oxide gas, hydrogen, propane, the butane, and described small capsule bubble diameter is distributed in micron and/or nanoscale.

On the bubble of capsule described in technique scheme medical diagnosis, be applied to ultrasonic contrast imaging as acoustic contrast agent.

At the capsule bubble of the oxygen of parcel described in the technique scheme, on clinical treatment, be applied to vein oxygen therapy.

Consider the safety and the cost of clinical practice, preferred version mainly select following four kinds through food and drug administration approval can safety as parenteral alimentation through intravenous hydrophilic lipophilic amphiphatic molecule table and activating agent as filmogen: lecithin, polysorbas20, Tween 80, Cremophor EL.With cholesterol as the film table and modification additives is allocated.By using, adopt the perfluoropropane gas of good stability as ultrasonic contrast.As the clinical practice of vein oxygen therapy, then adopt the oxygen parcel.Biodegradable water solublity powdery molecule carrier adopts the glucose crystal, and aqueous medium adopts normal saline.

The present invention is the purpose that reaches liquid phase core cavitation, adopted be different from conventional methods at 3: 1) at first in gel state to film forming on the liquid crystal state phase transition temperature on biodegradable water miscible powdery molecule carrier, because the particle size distribution of water miscible powdered granule is at micron order and/or nanoscale, be actually the liquid phase core solid state at first that liposome (Liposome) or non-ionic surface active corpusculum (Niosome) or both is mixed the phospholipid-non-ionic surface active mixing corpusculum of formation, help so simultaneously preserving and packing; 2) injecting aqueous medium in use, in gel state manually powerful in short-term concussion under the liquid crystal state phase transition temperature, is to avoid film phase transition process parcel liquid component under the high temperature.Under manually shaking, the dissolving of solidity sol particle core, film subsides when support particles is dissolved, and concussion simultaneously forms frequent liquid, film, gas contact interface, thereby gas enters the purpose that the interior also spontaneous sealing of film reaches liquid phase core cavitation, and the capsule bubble that forms is also abundant.3) the present invention adopts vacuum-gas displacement method, can make the liquid phase cavitation reach the purpose of specific gas cavitation, helps according to the selected specific gas of specific purposes.

By the present invention, with the small capsule bubble of liquid phase core fluoroalkane hydro carbons gaseous cavitation through intravenous injection, because diameter is less than 10 microns, can be smoothly by pulmonary circulation, can form stable ultrasonic contrast interface and reach the purpose of the left heart, the right heart and abdominal part parenchymal viscera radiography.

By the present invention, with the small capsule bubble of liquid phase core oxygen cavitation through venoclysis, simultaneously in conjunction with the complex that carries oxygen (porphyrin compound) of hemoglobin and synthetic infusion together.Because the liposome of core cavitation or claim the microbubble of film parcel, under the ultrasonic/sonic wave vibration, can cavitation take place and break.Therefore in the small capsule bubble of input liquid phase core oxygen cavitation, external, utilize ultrasonic probe in local searchlighting, local small capsule collapse of bubbles, can make on the one hand in the local blood capillary arterial blood partial pressure of oxygen far above partial pressure of oxygen in the tissue, can make oxygen-starved tissue obviously improve the disperse of oxygen on the other hand, thereby improve the oxygen supply of oxygen-starved tissue.Contain a large amount of generations that certain density active oxygen can suppress free radical, regulate metabolic defect in cellular calcium ion concentration, the human body immunity improving ability increases body to anoxybiotic tolerance, and can alleviate the tissue injury that is caused by ischemia-reperfusion.Can improve blood oxygen pressure and blood oxygen saturation rapidly; improve the tissue ischemia anaerobic condition; improve erythrocyte deformability, reduce hematoblastic cohesiveness, increase the fibrinolysis degree; regulate cell Ca++ ion concentration; the immunity and the anti-infection ability of effectively protection damaged tissue, and raising body are applicable to such as cerebrovascular disease; the myocardial ischemia pathological changes, acute cardiovascular and cerebrovascular disease patient's such as suffocate rescue and treatment.

The final preparation form of the present invention comprises three parts: the 1) container of certain volumetrical vacuum state, and it includes the biodegradable water solublity powdery molecule of the film parcel of certain mass; With 2) one comprise volumetrical airbag of specific gas or syringe, its volume equates with said vesse; With 3) syringe or a container that contains the aqueous medium of a constant volume.Final preparation form also can be reduced to two parts: 1) certain volumetrical container of having changed through vacuum-gas phase, the biodegradable water solublity powdery molecule that its film that includes certain mass wraps up and the container of specific gas; With 2) syringe or a container that contains the aqueous medium of a constant volume.

In sum, the small capsule bubble of the self-enclosed formation of hydrophilic lipophilic amphiphatic molecule of the liquid phase core cavitation of the present invention's preparation is compared with existing other microbubble acoustic contrast agents and is had following tangible advantage: 1) with low cost.The used lecithin of preferred version of the present invention is also cheap membrane material the most commonly used in the phospholipid surfactant; Tween 80, polysorbas20, glucose crystal and cholesterol have had a large amount of manufacturer production because of inside and outside, and product price is cheap, and are easy to buying; 2) safety non-toxic, but but therefore the hydrophilic lipophilic amphiphillic surfactant of intravenous injection that all material that the present invention adopts is the human body biological metabolism or has can be used as parenteral alimentation through food and drug administration approval has high safety as filmogen; 3) owing to adopt therapy in dry powder form to be easy to preparation and packing; And again through manually concussion dissolving, the capsule bubble of formation is the abundantest in use, and result of use is best; 3) the capsule bubble diameter is distributed in the 3-5 micrometer range, can be smoothly by pulmonary circulation, can reach the purpose of substantial viscera and heart radiography through vein or intra-arterial injection; 4) be easy to storage, room temperature just can keep all physics chemistry characteristic down; 5) it is remarkable to use its radiography effect as the clinical ultrasound diagnostic imaging, and the radiography length of holding time can be kept more than half an hour; 6) danger of no metallic particles pollution; 7), can be used for vein oxygen therapy as the clinical treatment purposes.

Description of drawings

Fig. 1 is the non-ionic surface active corpusculum 20 power microscope figure of the liquid phase core perfluoropropane gaseous cavitation of embodiment 3.

Fig. 2 is the normal distribution curve figure of non-ionic surface active corpusculum diameter of the computer image analysis core perfluoropropane gaseous cavitation of embodiment 3.

Fig. 3 is the liposome 20 power microscope figure of the general air cavitation of liquid phase core of embodiment 4.(arrow is represented the bubble that breaks among the figure)

Fig. 4 is the liposome of the core perfluoropropane gaseous cavitation of embodiment 5.

Fig. 5 is the microscope figure of the nonionic-phospholipid mixing corpusculum of the liquid phase core cavitation of embodiment 6.(not seeing disruptive capsule bubble among the figure)

Fig. 6 is the 20 power microscope figure of nonionic-phospholipid mixing corpusculum of the liquid phase core perfluoropropane gaseous cavitation of embodiment 7.

Fig. 7 is the normal distribution curve figure of the phospholipid-non-ionic surface active mixing corpusculum diameter of the computer image analysis core perfluoropropane gaseous cavitation of embodiment 7.

Fig. 8 A is a kidney energy diagram image before the zoopery radiography, kidney energy diagram image behind the 8B radiography.

Fig. 9 is the nonionic-phospholipid mixing corpusculum 20 power microscope figure of liquid phase core gaseous cavitation.

The specific embodiment

Describe performance of the present invention in detail below by embodiment and related experiment:

Embodiment 1: the non-ionic surface active corpusculum of preparation liquid phase core cavitation

At first 1 gram maltodextrin is ground to micron-sized powdered granule, class 60 of 100mg department is miscible in chloroform organic solution with the 20mg cholesterol, and in the aerosol apparatus of packing into.Introduce 50 milliliters of round bottom ground flasks, sparge on the maltodextrin, insert Rotary Evaporators, evacuation under 60 degree water-baths, rotary evaporation 30 minutes, film forming is on the maltodextrin powdered granule.Take out the maltodextrin that film covers, under the room temperature air-dry 12 hours.Above-mentioned film parcel maltodextrin is dissolved in rapidly in 10 ml physiological salines, manually vibrated 30 seconds, get suspension and observe in microscopically, core cavitation non-ionic surface active corpusculum diameter is about the 7-8 micron, and a high power field distributes about 90%.At room temperature, under the packing less situation, small capsule bubble can keep not disappearing and breaking in 3 days in flask: under the sealing state, small capsule bubble can keep at least 1 week.

Embodiment 2: the non-ionic surface active corpusculum of allotment HLB value preparation liquid phase core cavitation

1 gram maltodextrin is ground to micron-sized powdered granule.It is 6 compound emulsifying agents that the HLB value of department class 80 and Tween 80 is mixed the HLB value by HLB value weight average computation separately, wherein class of department 80 (166mg) weight accounts for 83%, Tween 80 (34mg) weight accounts for 17%, above-mentioned compound emulsifying agent and 20mg cholesterol is miscible in chloroform organic solution, and in the aerosol apparatus of packing into.Introduce 50 milliliters of round bottom ground flasks, sparge on the maltodextrin, insert Rotary Evaporators, evacuation under 60 degree water-baths, rotary evaporation 30 minutes, film forming is on the maltodextrin powdered granule.Take out the maltodextrin that film covers, under the room temperature air-dry 12 hours.Above-mentioned film parcel maltodextrin is dissolved in rapidly in 10 ml physiological salines, gets suspension and observe in microscopically, core cavitation non-ionic surface active corpusculum diameter is about the 7-8 micron, and a high power field distributes about 90%.At room temperature, under the packing less situation, small capsule bubble can keep not disappearing and breaking in 1 day in flask.Under the sealing state, small capsule bubble can keep at least 1 week.

Embodiment 3: the non-ionic surface active corpusculum of allotment HLB value preparation liquid phase core perfluoropropane gaseous cavitation

1 gram maltodextrin is ground to micron-sized powdered granule.It is 6 compound emulsifying agents that the HLB value of department class 80 and Tween 80 is mixed the HLB value by HLB value weight average computation separately, wherein class of department 80 (166mg) weight accounts for 83%, Tween 80 (34mg) weight accounts for 17%, above-mentioned compound emulsifying agent and 20mg cholesterol is miscible in chloroform organic solution, and in the aerosol apparatus of packing into.Introduce 50 milliliters of round bottom ground flasks, sparge on the maltodextrin, insert Rotary Evaporators, evacuation under 60 degree water-baths, rotary evaporation 30 minutes, film forming is on the maltodextrin powdered granule.Take out the maltodextrin that film covers, under the room temperature air-dry 12 hours.Above-mentioned film parcel maltodextrin is sub-packed in the vial extracting vacuum, and sealing.Inject the gas (gas volume is consistent with aforementioned vessel volume) and 10 ml physiological salines of perfluoropropane sequentially respectively, manually concussion is after 30 seconds, getting suspension observes in microscopically, the non-ionic surface active corpusculum diameter of core perfluoropropane gaseous cavitation is about the 3-8 micron, it is about 95% that high power field distributes, and sees Fig. 1, is finding under 20 times of mirrors, 95% small vesicle diameter is about the 7-8 micron, and only a small amount of capsule bubble diameter is a bit larger tham 10 microns.Fig. 2 is the normal distribution curve figure of non-ionic surface active corpusculum diameter of the computer image analysis core perfluoropropane gaseous cavitation of embodiment 3.By the normal curve scattergram as seen, the diameter of 65% capsule bubble distributes at the 2-6 micrometer range, and 99% capsule bubble is less than 10 microns.Average capsule bubble diameter is 4 microns.Adopt the capsule bubble stability of perfluoropropane gaseous cavitation to improve greatly, at the blow-by state, small capsule bubble can be kept 3-4 days, and the sealing state small capsule bubble of parcel perfluoropropane down keeps January at least.

Embodiment 4: the liposome of preparation liquid phase core cavitation

100 milligrams of lecithin and 40 milligrams of cholesterol are miscible in 5 milliliters of chloroform organic solutions, and sparge 1 gram glucose micro crystal in the aerosol apparatus of packing into, and insert 50 milliliters of round bottom ground flasks, insert Rotary Evaporators, evacuation under the 60 degree water-baths, the rotary evaporation film forming is in drag.After 30 minutes, take out film and cover the glucose micro crystal, under the room temperature air-dry 12 hours.Above-mentioned film parcel maltodextrin is dissolved in rapidly in 10 ml physiological salines, and manually strong vibration is got suspension and is observed in microscopically, and the liposome diameter of core cavitation is about the 3-8 micron, and a high power field distributes about 90%.Under the room temperature, keep open-ended, small capsule bubble was held time about 6 hours.But the liposome structure of the core cavitation of simple lecithin bilayer shows stable inadequately, is easy to break.Fig. 3 is a finding under liposome 20 power microscopes of the general air cavitation of liquid phase core of embodiment 4, this is a finding under 20 times of mirrors, 90% small vesicle diameter is about 5 microns, only a small amount of capsule bubble diameter is a bit larger tham 10 microns, but the liposome structure of the core cavitation of simple lecithin bilayer shows stable inadequately, be easy to break, see arrow indication among Fig. 3.

Embodiment 5: the liposome of preparation liquid phase core perfluoropropane gaseous cavitation

100 milligrams of lecithin and 40 milligrams of cholesterol are miscible in 5 milliliters of chloroform organic solutions, and sparge 1 gram glucose micro crystal in the aerosol apparatus of packing into, and insert 50 milliliters of round bottom ground flasks, insert Rotary Evaporators, evacuation under the 60 degree water-baths, the rotary evaporation film forming is in drag.After 30 minutes, take out film and cover the glucose micro crystal, under the room temperature air-dry 12 hours.Above-mentioned film parcel glucose micro crystal is sub-packed in the vial extracting vacuum, and sealing.Inject the gas (gas volume is consistent with aforementioned vessel volume) and 10 ml physiological salines of perfluoropropane sequentially respectively, powerful concussion is after 30 seconds, getting suspension observes in microscopically, the liposome diameter of core perfluoropropane gaseous cavitation is about the 3-8 micron, it is about 95% that a high power (20 *) visual field distributes, and stability significantly improves, under the blow-by state, small capsule bubble can keep about 3-4 days.The sealing state small capsule bubble of parcel perfluoropropane down keeps January at least.Fig. 4 be the liposome diameter of core perfluoropropane gaseous cavitation of embodiment 5 about the 3-8 micron, it is about 95% that high power field distributes, and do not see that disruptive capsule bubble stability significantly improves in the full visual field.

Embodiment 6: the phospholipid-non-ionic surface active mixing corpusculum of preparation liquid phase core cavitation

100 milligrams of lecithin, 50 milligrams of Tween 80s, 30 milligrams of cholesterol are miscible in 5 milliliters of chloroform organic solutions, and sparge 1 gram glucose micro crystal in the aerosol apparatus of packing into, and insert 50 milliliters of round bottom ground flasks, insert Rotary Evaporators, evacuation under the 60 degree water-baths, the rotary evaporation film forming is in drag.After 30 minutes, take out film and cover the glucose micro crystal, under the room temperature air-dry 12 hours.Above-mentioned film parcel glucose micro crystal is placed 10 ml physiological salines rapidly, manually strong vibration, get suspension and observe in microscopically, the phospholipid of core cavitation-non-ionic surface active mixing corpusculum diameter is about the 3-8 micron, and a high power field distributes about 90%.Only wrap up the nonionic phospholipid body structure of the liquid phase core cavitation of general air and stablize, obviously be better than the capsule bubble of embodiment 4 simple lecithin bilayers parcels.Under the room temperature, keep open-ended, small capsule bubble was held time about 2-3 days, and sealing state can keep at least 1 week.See Fig. 5.Fig. 5 is microscope figure below of the nonionic-phospholipid mixing corpusculum of the liquid phase core cavitation of embodiment 6, and the phospholipid of core cavitation-non-ionic surface active mixing corpusculum diameter is about the 3-8 micron, and a high power (20 *) visual field distributes about 90%.Do not see disruptive capsule bubble.

Embodiment 7: the phospholipid-non-ionic surface active mixing corpusculum of preparation liquid phase core perfluoropropane gaseous cavitation

100 milligrams of lecithin, 20 milligrams of Tween 80s, 30 milligrams of cholesterol are miscible in 5 milliliters of chloroform organic solutions, and sparge 1 gram glucose micro crystal in the aerosol apparatus of packing into, and insert 50 milliliters of round bottom ground flasks, insert Rotary Evaporators, evacuation under the 60 degree water-baths, the rotary evaporation film forming is in drag.After 30 minutes, take out film and cover the glucose micro crystal, under the room temperature air-dry 12 hours.Above-mentioned film parcel glucose micro crystal is sub-packed in the vial extracting vacuum, and sealing.Inject the gas (gas volume is consistent with aforementioned vessel volume) and 10 ml physiological salines of perfluoropropane sequentially respectively, powerful concussion is after 30 seconds, getting suspension observes in microscopically, the phospholipid of core perfluoropropane gaseous cavitation-non-ionic surface active mixing corpusculum diameter is about the 3-5 micron, a high power field distributes about 95%, stability significantly improves, and sees Fig. 6, Fig. 7.Stability significantly improves, and under the blow-by state, small capsule bubble can keep about 1 week.The sealing state small capsule bubble of parcel perfluoropropane down keeps February at least.Fig. 6 is microscope figure below of nonionic-phospholipid mixing corpusculum of the liquid phase core perfluoropropane gaseous cavitation of embodiment 7, the phospholipid of core perfluoropropane gaseous cavitation-non-ionic surface active mixing corpusculum diameter is about the 3-5 micron, it is about 95% that high power field distributes, and stability significantly improves.Fig. 7 is the normal distribution curve figure of the phospholipid-non-ionic surface active mixing corpusculum diameter of the computer image analysis core perfluoropropane gaseous cavitation of embodiment 7.By the normal curve scattergram as seen, the diameter of 70% capsule bubble distributes at the 2-6 micrometer range, and 97% capsule bubble is less than 10 microns.Average capsule bubble diameter is 4 microns.

Embodiment 8: the ultrasonic contrast zoopery

Zoopery is injected 1 milliliter of the suspension of experiment 7 prepared liquid phase core cavitations in the White Rabbit body through rabbit ear edge vein group, and in the several seconds, under the color doppler power imaging imaging pattern, visible significant radiography reinforced effects is seen Fig. 8 A, 8B.Fig. 8 A is a kidney energy diagram image before the radiography, and Fig. 8 B is a kidney energy diagram image behind the radiography, and the video picture of excess of the kidney matter significantly strengthens.

Embodiment 9: the phospholipid-non-ionic surface active mixing corpusculum of preparation liquid phase core perfluoropropane gaseous cavitation

100 milligrams of lecithin, 5 milligrams of Cremophor EL, 30 milligrams of cholesterol are miscible in 5 milliliters of chloroform organic solutions, and sparge 1 gram glucose micro crystal in the aerosol apparatus of packing into, and insert 50 milliliters of round bottom ground flasks, insert Rotary Evaporators, evacuation under the 60 degree water-baths, the rotary evaporation film forming is in drag.After 30 minutes, take out film and cover the glucose micro crystal, under the room temperature air-dry 12 hours.Above-mentioned film parcel glucose micro crystal is sub-packed in the vial extracting vacuum, and sealing.Inject the gas (gas volume is consistent with aforementioned vessel volume) and 10 ml physiological salines of perfluoropropane sequentially respectively, powerful concussion is after 30 seconds, getting suspension observes in microscopically, the phospholipid of core perfluoropropane gaseous cavitation-non-ionic surface active mixing corpusculum diameter is about the 7-8 micron, and a high power field (20 *) distributes about 85%.

Embodiment 10: the phospholipid-non-ionic surface active mixing corpusculum of preparation liquid phase core oxygen gas cavitation

100 milligrams of lecithin, 20 milligrams of Tween 80s, 30 milligrams of cholesterol are miscible in 5 milliliters of chloroform organic solutions, and sparge 1 gram glucose micro crystal in the aerosol apparatus of packing into, and insert 50 milliliters of round bottom ground flasks, insert Rotary Evaporators, evacuation under the 60 degree water-baths, the rotary evaporation film forming is in drag.After 30 minutes, take out film and cover the glucose micro crystal, under the room temperature air-dry 12 hours.Above-mentioned film parcel glucose micro crystal is sub-packed in the vial extracting vacuum, and sealing.Inject the gas (gas volume is consistent with aforementioned vessel volume) and 10 ml physiological salines of perfluoropropane sequentially respectively, powerful concussion is after 30 seconds, getting suspension observes in microscopically, the phospholipid of core oxygen gas cavitation-non-ionic surface active mixing corpusculum diameter is about the 3-5 micron, a high power field distributes about 95%, under the blow-by state, small capsule bubble can keep about 1 week.The sealing state small capsule bubble of parcel oxygen down keeps February at least.

Embodiment 11: the phospholipid-non-ionic surface active mixing corpusculum of allotment HLB value preparation liquid phase core gaseous cavitation

Powdered granule with 1 gram glucose.It is 9 compound emulsifying agent that the HLB value of lecithin and polysorbas20 is mixed the HLB value by HLB value weight average computation separately, wherein polysorbas20 (22mg) weight accounts for 11%, lecithin (178mg) weight accounts for 89%, above-mentioned compound emulsifying agent and 20mg cholesterol is miscible in chloroform organic solution, and in the aerosol apparatus of packing into.Introduce 50 milliliters of round bottom ground flasks, sparge on the glucose, insert Rotary Evaporators, evacuation under 60 degree water-baths, rotary evaporation 30 minutes, film forming is on glucose powdery granule.Take out the glucose that film covers, under the room temperature air-dry 12 hours.Above-mentioned film parcel glucose is dissolved in rapidly in 10 ml physiological salines, manually powerful concussion is after 10 seconds, get suspension and observe in microscopically, core cavitation phospholipid-non-ionic surface active mixing corpusculum diameter is about the 5-6 micron, but air bubble content obviously is less than and Tween 80 allotment gained.As Fig. 9, shown in, small capsule bubble diameter obviously reduces but compare air bubble content with embodiment 6,7 all at the 5-6 micron.

Embodiment 12: through vein oxygen therapy zoopery

Get 5 milliliters of the suspensions of phospholipid-non-ionic surface active mixings corpusculum of the oxygen core cavitation of embodiment 10 preparation, employing is through the mode of White Rabbit auricular vein infusion, with the speed infusion of 0.05 milliliter of per minute.In 20 minutes infusion process, do not see any gas embolism sign, obviously improved the Sanguis Leporis seu oryctolagi oxygen content.

Need to prove: to those of ordinary skill in the art; under the prerequisite that does not change the principle of the invention, can also make some changes or distortion to the present invention; some specific antibody of labelling or wrap up special ingredient on filmogen for example; to reach the purpose of targeting release, targeting radiography, targeted therapy, this belongs to protection scope of the present invention equally.

Claims

1. A method for cavitating the liquid-phase core of a hydrophilic-lipophilic self-sealing corpuscle into a vesicle, wherein the hydrophilic-lipophilic amphiphilic self-sealing corpuscle is composed of cholesterol and one selected from nonionic surfactants and phospholipids Composition, or composed of cholesterol, non-ionic surfactants, phospholipids;

It is characterized in that the method comprises the steps of:

(1) The film-forming material is dissolved in chloroform and/or methanol, and spray-dried at the phase transition temperature from the gel state to the liquid crystal state to form a film on a biodegradable water-soluble tiny powder particle carrier in a vacuum state , the mass ratio of the film-forming material to the water-soluble carrier is 1:50-500000; the particle size distribution of the biodegradable water-soluble powdery microparticle carrier and the film-wrapped biodegradable water-soluble powdery microparticle carrier is within a micron scale and/or nanoscale;

(2) Put the above-mentioned water-soluble tiny powdery particles wrapped in a film into a container with a certain volume, put it in a vacuum, and seal it, and inject a certain volume in order at a temperature lower than the phase transition temperature from the gel state to the liquid crystal state. The gas and water-based medium are shaken manually until the water-soluble tiny powder particles are dissolved, and the envelope collapses to allow the gas to enter the self-closed body of the hydrophilic-lipophilic amphiphilic molecule, thereby forming a liquid-phase core cavitation of the hydrophilic-lipophilic amphiphilic molecule. Closed corpuscular vesicles.

2. The method according to claim 1, wherein the liquid-phase core of hydrophilic-lipophilic self-sealing corpuscles is cavitated into vesicles, characterized in that the self-sealing corpuscles of hydrophilic-lipophilic amphiphilic molecules that are cavitated by the liquid-phase core refer to Nonionic surface active bodies composed of nonionic surfactants and cholesterol, or liposomes composed of cholesterol and phospholipids or lipid nonionic surface active mixed bodies composed of cholesterol, nonionic surfactants and phospholipids.

3. A method for cavitation of the liquid-phase core of hydrophilic and lipophilic self-enclosed corpuscles into vesicles according to claim 1 or 2, characterized in that the non-ionic surface-active corpuscles use the Span series non-ionic surface Active agent, one or more of Tween series non-ionic surfactants, Brij non-ionic surfactants, Cremophor EL, Pluronic F68, albumin, and cholesterol membrane surface additives.

4. A method for cavitating the liquid-phase core of hydrophilic and lipophilic self-enclosed corpuscles into vesicles according to claim 2, characterized in that the phospholipids used in the liposomes are selected from the following types: One or more of them are formulated and composed with cholesterol-based membrane surface additives; the phospholipid-type hydrophilic and lipophilic amphiphilic molecules are lecithin, phosphatidylethanolamine, cholesterol, cholesterol acetyl ester, β-sitosterol, ox bile sodium phosphate, egg phosphatidylcholine, phosphatidylinositol, sphingomyelin, sphingomyelin, dicetyl acetophosphatidylcholine, myristoyl lecithin, stearylamide, oleic acid monophospholipid, vitamin E, 1,2-palm Acyl-sn-glyceryl-3-phosphatidic acid glyceryl-sodium salt, 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glyceryl - 3-phosphatidic glyceric acid sodium salt, 1,2-palmitoyl-sn-glyceryl-3-3-phosphatidylcholine or mixtures of the above compounds.

5. A method for cavitating the liquid-phase core of hydrophilic-lipophilic self-enclosed corpuscles into vesicles according to claim 2, characterized in that the lipid non-ionic surface active mixed corpuscles, the phospholipids of which are hydrophilic and hydrophilic The ratio of lipid amphiphilic molecules and non-ionic surfactants to hydrophilic and lipophilic amphiphilic molecules is 1-500:1.

6. A method for cavitating the liquid-phase core of hydrophilic-lipophilic self-enclosed bodies into vesicles according to claim 1, characterized in that the biodegradable water-soluble powdery microparticle carrier is any of the following or Several mixtures: polylactic acid, polylactide, polycaprolactone, polyglycolic acid, polyhydroxybutyric acid, polyvaleric acid, polycapric acid, polylactic acid and polyglycolic acid copolymer PLGA, polylactic acid and poly Copolymer of hydroxybutyric acid, polyisohexyl cyanoacrylate, polylactide glycolide, polyanhydride, semi-solid polyorthoester, polyethylene glycol, polyvinylpyrrolidone, dibutylhydroxytoluene, sorbitol, Sodium carboxymethylcellulose, hydroxyethyl starch, sodium carboxymethyl starch, alginate, glucose, galactose, fructose, maltose, sucrose, maltodextrin, dextran, water-soluble chitosan, sodium chloride .

7. A method for cavitating the liquid-phase core of hydrophilic-lipophilic self-enclosed corpuscles into vesicles according to claim 1, characterized in that the injection gas is fluoroalkane gas, air, oxygen, nitrogen, carbon dioxide Gas, nitrogen monoxide, hydrogen, propane, butane, or any one or more of them, and the diameter of the vesicle bubbles is distributed in the micron and/or nanometer scale.