CN101711736A - Method for preparing medicine-carrying microvesicle - Google Patents
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Abstract
The invention relates to the field of medicines, in particular to a medicine-carrying microvesicle and a preparation method thereof. In the method, medicine-carrying high molecular polymer class nano particles are connected with a lipid microvesicle by an amido bond so as to prepare the medicine-carrying microvesicle. Under an ultrasonic action, the lipid microvesicle of the medicine-carrying microvesicle is broken, and the medicine-carrying high molecular polymer class nano particles which are connected with the lipid microvesicle of the medicine-carrying microvesicle are released and enter the clearances of tissues to exert an action. The method has mild reaction conditions and superior chemical selectivity of the reaction process; and the prepared medicine-carrying microvesicle can be used as an ultrasonic contrast agent, can also be used as a controlled release carrier for various medicines and has extensive application prospect.
Description
Technical field
The present invention relates to field of medicaments, be specifically related to a kind of preparation method of medicine carrying microvesicle.
Background technology
Tumor is one of three big diseases of current serious threat human health.The conventional chemotherapy medicine of extensive use in clinical cancer therapy at present mostly is non-selective medicine, in the kill tumor cell, also can kill normal cell, conventional therapy dosage can produce significant toxic and side effects to normal histoorgan, causes the patient not tolerate, and reduces curative effect of medication.Therefore, the key of antitumor drug research and development is to improve the tumor-selective of medicine, reduces its gathering at non-targeting moiety.
In recent years, along with continuing to bring out of subject development, infiltration and new techniques such as molecular pharmacology, biopharmaceutical analysis, cell drug chemistry, drug molecule transmission and system analysis engineering, pharmaceutical dosage form and preparation research have entered targeting drug delivery system (Targeted drug deliverysystem, TDDS) epoch.Targeted drug treatment makes medicine aiming tumor locus, preserves higher relatively concentration in the part, prolong drug action time improves the lethality to tumor cell, but less to the normal tissue cell effect.At present, the medicine that is used for neoplasm targeted therapy has control sustained-release chemotherapy medicine, liposome chemotherapeutic, molecular targeted agents, radionuclide etc., and medicine can give by multipath, as blood vessel insertion administration, ultrasonic mediation targeting percutaneous dosing etc.
Nano-medicament carrier in the control slow releasing pharmaceutical is one of focus of present tumor-targeting drug preparation research.Nano-medicament carrier can improve the drug level of target area, thereby improves the utilization rate and the curative effect of medicine, reduces the untoward reaction of medicine.Nano microsphere has following advantage at medicine and gene transporter mask: but 1. slow releasing pharmaceutical improves blood drug level, prolong drug action time; 2. can reduce drug degradation, improve medicine stability; 3. can protect nucleotide, prevent that it is by nuclease degradation; 4. can improve the nucleotide transfection efficiency; 5. can set up new route of administration.
The targeted nano medicine microspheres is linked to each other with the Nano medication microsphere by targeted molecular and forms, and the intrinsic advantage, also has more than possessing the Nano medication microball preparation: the purpose that 1. can reach targeted; 2. can further reduce or avoid the advantages such as toxic and side effects of medicine guaranteeing to reduce dosage under the pharmaceutically-active prerequisite.Yet the problem of targeted nano drug development still has a lot, mainly comprises following several respects: 1. medicine parcel efficient is lower: 2. the biocompatibility of nano material is still waiting to improve; 3. the joint efficiency of targeting molecule and Nano medication is lower and be connected back targeting molecular activity and reduce: 4. because the complexity and the existing various barrier thereof of human body cause Nano medication not high to the targeting of tumor.
(Ultrasound-targeted microbubbledestruction, UTMD) mediation drug targeting treatment technology is a kind of target administration new method that has potentiality that developed recently gets up in ultrasonic targeted microbubble explosion.This method is made up of medicine carrying microvesicle system and localization by ultrasonic controlled release system two parts.Its principle be with the medicine carrying microvesicle after intravenous injection, adopt the ultrasonic irradiation of certain energy through body surface, make microvesicle locate explosion in vivo and discharge medicine, simultaneously, " acoustic horn effect " that the microvesicle explosion is produced (sonoporation) can make local blood capillary and permeability of cell membrane increase again, promote the infiltration of medicine, locate the purpose that discharges and improve therapeutic effect in the medicine body thereby reach.Domestic and international research shows, UTMD mediation drug targeting treatment technology can effectively improve the treatment local drug concentration, improves therapeutic effect, reduce toxic and side effects, but still have some technical barriers, mainly show following three aspects: 1. microvesicle is as pharmaceutical carrier, its drug loading deficiency.Because the center of microvesicle is a noble gas, shell is poor adipose membrane, limited by the microvesicle preparation method, can't realize pharmaceutical pack is entrained in the microvesicle, its drug loading of medicine carrying microvesicle of currently used surface adhesion method (medicine is directly adhered to the microvesicle surface) or integration method (medicine is dissolved in the microvesicle plasma membrane) preparation is all very limited; 2. low, the weak point of holding time of medicine local concentration.The medicine that carry on the microvesicle surface is a property crossed in partial concentration after discharging with the microvesicle explosion, and the weak point of holding time is difficult to reach the treatment requirement; 3. targeting remains further to be improved.
In recent years, the research of relevant ultrasound microbubble contrast agent has obtained continuous progress, and obtain many related invention patents, as: a kind of ultrasound microbubble contrast agent (patent No.: ZL 200410021905.3) that carries gene, carry the ultrasound microbubble contrast agent and preparation method thereof (patent No.: ZL 200510127996.3) of gene or medicine etc., and the microvesicle that adopts macromolecular material to make comes with some shortcomings, for example its shell is harder, the elasticity relative mistake, a little less than the cavitation effect in ultrasonic field, can not can be caused normal tissue injury again if strengthen ultrasonic energy by low-yield ultrasonic smashing, the ultrasonoscopy effect is not so good as lipid microbubble etc. in the body.Though and lipid microbubble has stronger cavitation in ultrasonic field, can be broken up by more low-yield ultrasonic irradiation, but also there are some defectives in lipid microbubble, for example: its bag medicine carrying thing instability, medicine normally is attached on the lipid microbubble surface or is inlaid into the parietal layer of filmogen phospholipid, and the amount and the character that enter back medicine in the body may be subjected to influence of various factors in the body; Simultaneously because its kernel is a gas, so entrapment efficiency and drug loading are not high; In addition, lipid microbubble is generally micron order, can not penetrate the tumor vascular endothelium gap and enters tumor tissues.
Summary of the invention
Purpose of the present invention can not penetration rate of blood endothelial tube gap, entrapment efficiency at the prior art lipid microbubble and drug loading is not high, short defective of half-life in the body, and a kind of preparation method of novel medicine carrying microvesicle is provided.
For achieving the above object, technical scheme provided by the present invention is:
A kind of preparation method of medicine carrying microvesicle may further comprise the steps:
Step 1: distearoyl phosphatidylcholine, another kind of phospholipid, glycerol are mixed with phosphate buffer and heat, the mass ratio of described distearoyl phosphatidylcholine and another kind of phospholipid is 4-6: 2, the volume ratio of glycerol and PBS is 1: 2-4, and the quality summation of DPPC and another kind of phospholipid is counted 3-5 with the ratio of the volume summation of glycerol and PBS with mg/ml: 1; Charge into perfluoropropane gas or HFC-236fa gas vibration 40-50s, standby;
Step 1 enters the gas parcel in the microvesicle by machinery concussion method, and the lipid microbubble hollow of preparation includes fluorocarbon gas, its diameter Distribution scope 2-6um, and the half-life is 25-35 minute.
Step 2: be dissolved in the 1ml dichloromethane containing the macromolecule polymeric material 40-50mg that holds carboxyl in the molecule side group, add 200-250 μ l medicine, the sound 35-45s that shakes gets colostrum; Colostrum is mixed with 3-6ml 5%-7% poly-vinyl alcohol solution,, get emulsion with 20000-23000r/min high speed dispersion homogenizing effect 5min, add 8-15ml 2%-4% isopropyl alcohol again, stir 2-5h, after rinsing, it is standby to be the medicine-carrying polymer polymer nanoparticle after the lyophilization; Wherein the concentration of poly-vinyl alcohol solution and isopropyl alcohol is the mass volume ratio in g/ml.
The medicine-carrying polymer polymer nanoparticle of step 2 preparation also can adopt the additive method preparation, referring to document:
Lv Guixiang, Peng Haisheng, Liu Xijun etc. the preparation of voriconazole PLGA nanoparticle and form thereof and release feature analysis. the Chinese biological goods are learned magazine, 2008,21 (1): 42-44;
Hu Yunxia, former continuous ripple, Zhang Xiaojin etc. the finishing of polylactic acid drug-carried nanometer and in-vitro evaluation. Chinese biological engineering in medicine journal, 2004,23 (1): 30-36.
Step 3: add positively charged biochemical reagents in step 1 gained solution, the constant temperature vibration is hatched, and the lipid microbubble of leaving and taking the upper strata is standby; This step makes lipid microbubble surface charge-modified of step 1 gained.
Step 4: it is that 0.1M, pH are in 6.0 the buffer solution that the medicine-carrying polymer polymer nanoparticle that N-hydroxy thiosuccinimide and another kind of carboxyl activator and step 2 are made fully is mixed in concentration, incubation reaction 30-80min under the room temperature, centrifugal rinsing, get the precipitation standby; Described centrifugal rinsing is in order to remove unreacted carboxyl activator.This step makes the activated carboxylic on medicine-carrying polymer polymer nanoparticle surface.
Step 5: it is that 0.1M, pH are in 8.0 the buffer solution that the lipid microbubble of step 3 gained and step 4 gained precipitation fully are mixed in concentration, 0 ℃-4 ℃ hatch 25-40min with 30-45r/min vibration after, be transferred to 4 ℃ and leave standstill and hatch 24-72h, leave and take the microvesicle on upper strata.
Preferably, the described phospholipid of step 1 is DSPE-PEG (2000)-NH2, described being heated to be at 35 ℃-40 ℃ heating 30-40min.
Preferably, the described macromolecule polymeric material of step 2 is a kind of among polylactic acid, the polylactic acid-glycolic guanidine-acetic acid copolymer p LGA.
Preferably, positively charged biochemical reagents are poly-l-lysine PLL described in the described step 3, and molecular weight is 3-7 ten thousand, and addition is 1.5-3.5mg.
Preferably, described step 30 ℃-4 ℃ of temperature with 30-45r/min vibration 25-40min.
Preferably, carboxyl activator is 1-(3-dimethyl propyl)-3-ethyl carbodiimide or 1-(3-dimethyl propyl)-3-ethyl-carbodiimide hydrochloride in the described step 4.
Preferably, N-hydroxy thiosuccinimide described in the described step 4 with the mol ratio of the mol ratio of medicine-carrying polymer polymer nanoparticle, another kind of carboxyl activator and medicine-carrying polymer polymer nanoparticle all greater than 30: 1.
Preferably, the described centrifugal speed of described step 4 is 3000-3500r/min, centrifugal 5min.
Preferably, buffer solution described in step 4, the step 5 is a kind of in PBS (phosphate buffer), MES (2-(N-morpholino) ethyl sulfonic acid) buffer, borate buffer solution, Tris (Tris) buffer.
The present invention also provides the medicine carrying microvesicle according to described preparation method preparation, and described medicine carrying microvesicle is made up of lipid microbubble and medicine-carrying polymer polymer nanoparticle, and described medicine-carrying polymer polymer nanoparticle is connected with lipid microbubble by amido link.
Preferably, the particle diameter of described medicine-carrying polymer polymer nanoparticle is 550-650nm.
The medicine carrying microvesicle of the method for the invention preparation is to connect the hybrid system that the high molecular polymer nanoparticle forms by the lipid microbubble surface, and it has the advantage of lipid microbubble and high molecular nanometer grain concurrently.Intravenous injection medicine carrying microvesicle of the present invention, when it arrives tumor locus, adopt this novel medicine carrying microvesicle of low-yield ultrasonic irradiation, can strengthen its cavitation effect, impel lipid microbubble to break, discharge the high molecular nanometer grain that its surface connects, the acoustic horn effect that produces when lipid microbubble breaks simultaneously can impel the of short duration opening in tumor neogenetic blood vessels endothelium gap, be beneficial to the high molecular nanometer grain and penetrate tumor neogenetic blood vessels endothelium gap, enter the slow medicine that discharges in the tumor tissues, to reach the purpose that suppresses tumor.High molecular nanometer grain size of the present invention is at nanoscale, can pass tumor neogenetic blood vessels endothelium gap, and the high molecular nanometer grain has the characteristic of slow release long-acting, its kernel is a liquid, and non-pneumatic, thereby can wrap the medicine carrying thing more efficiently and carry out sustained-release and controlled release.
The present invention is connected the lipid microbubble surface with medicine-carrying polymer polymer class nanoparticle by amido link, prepares a kind of novel medicine carrying microvesicle, both can be used as acoustic contrast agent, can be used as the controlled release carrier of various medicines again, has tumor-targeting.The method of the invention reaction condition gentleness, the course of reaction chemo-selective is superior.
The medicine carrying microvesicle explosion of accurate location, the medicine carrying microvesicle of efficient stable and the long as far as possible local active drug concentration of treatment of keeping in vivo is the key technology that transmit UTMD mediation medicine location.The medicine carrying microvesicle of the efficient stable of the method for the invention preparation, fundamentally solve the key technology difficult problem that microvesicle is low as medicine location its medicine carrying deficiency of release vehicle, local drug concentration, hold time short, the maturation and the clinical expansion of treatment basic research of tumour medicine targeting and UTMD mediation medicine location transmission technology had very far-reaching strategic importance.
Description of drawings:
Fig. 1 is the common light microscopic image (* 400) of the lipid microbubble of the method for the invention step 1 preparation;
Fig. 2 is the common light microscopic image of PLGA nanoparticle (* 400) that carries hemoporphyrin of embodiment 1 preparation;
Fig. 3 is the common light microscopic image of medicine carrying microvesicle (* 400) of embodiment 1 preparation;
Fig. 4 is the lipid microbubble light microscopic image (* 400) of embodiment 3 preparations;
Fig. 5 is the lipid microbubble light microscopic image (* 400) of embodiment 4 preparations;
Fig. 6 is the external release curve of the medicine-carrying polymer polymer nanoparticle of embodiment 1 and the preparation of embodiment 2 steps 2.
The specific embodiment:
Below in conjunction with the specific embodiment the present invention is described in further detail, but content of the present invention be not limited to for embodiment.
Embodiment 1
(1) preparation of common lipid microbubble:
With mass ratio respectively 2: 1 DPPC (distearoyl phosphatidylcholine) and DSPE-PEG (2000)-NH
2, volume ratio is that 1: 2 glycerol and PBS buffer adds in the plastics tubule and mix DPPC and DSPE-PEG (2000)-NH
2The quality summation count 6: 1 with the ratio of the volume summation of glycerol and PBS with mg/ml, 35 ℃ of heating in water bath with the air in the perfluoropropane gas instead tubule, carried out making lipid microbubble behind the mechanical oscillation 40s with argental mercury capsule dispenser after 30 minutes.The form of common lipid microbubble distributes and sees Fig. 1.As can be seen from Fig. 1, common lipid microbubble particle diameter is even, good dispersion degree.
(2) preparation of medicine-carrying polymer polymer nanoparticle:
40mg PLGA (for the PLGA of band carboxyl, molecular weight 25000, polymerization ratio 50: 50) is dissolved in the 1ml dichloromethane as oil phase, adds 200 μ l sensibion aqueous solutions in oil phase, the 35s that shakes gets colostrum.With colostrum and 3ml 5%PVA (polyvinyl alcohol, molecular weight is 30000-70000) solution mixes the back with 23000r/min homogeneous dispersion 5min, get emulsion, pour 8ml 2% isopropyl alcohol again into, the room temperature lower magnetic force stirs 2h, through centrifugal, distilled water rinsing 3 times is stored in behind the lyophilization 24h in 4 ℃ of refrigerators again.Fig. 2 is seen in the form and the distribution of the PLGA nanoparticle (HP-PLGA) of parcel hemoporphyrin.Common light microscopic is observed down, and medicine-carrying polymer nanoparticle particle size distribution is even, good dispersion degree, no adhesion and gathering.Fluorescence microscope is observed down, and the hemoporphyrin that parcel is gone in the nanoparticle sends bright red fluorescence certainly.
(3) the lipid microbubble surface is charge-modified:
In the common lipid microbubble solution that step 1 makes, add 0.5ml poly-D-lysine (PBS is mixed with the solution that concentration is 1.5-2.5mg/ml), 25min is hatched in 0 ℃ of vibration in the constant temperature oscillator, reuse PBS buffer (PH7.4) rinsing 3 times repeatedly, abandon subnatant, keep the lipid microbubble on upper strata, thereby make the positively charged lipid microbubble in surface.
(4) activated carboxylic on medicine-carrying polymer polymer nanoparticle surface
The PLGA nanoparticle of the parcel hemoporphyrin that carboxyl activator 1-(3-dimethyl propyl)-3-ethyl carbodiimide EDC, N-hydroxy thiosuccinimide sulfo-NHS and step 2 are made fully is mixed in the MES buffer of PH6.0 at 30: 30: 1 according to mol ratio, incubation reaction 30min under the room temperature, activation nanoparticle surface with carboxyl.Unreacted carboxyl activator is removed in the centrifugal rinsing of distilled water 3-5 time.
(5) coupling of lipid microbubble and medicine-carrying polymer polymer nanoparticle
The PLGA nanoparticle that lipid microbubble that step 3 gained is positively charged and step 4 gained have activated carboxyl fully is mixed in the MES buffer of PH8.0, and the 30min that vibrates in 4 ℃ of constant temperature oscillators moves into 4 ℃ of refrigerators again and leaves standstill and hatch 48h.Subnatant is abandoned in distilled water rinsing 3-5 time repeatedly, keeps the microvesicle that floats on the upper strata, promptly obtains the novel medicine carrying microvesicle that the surface is connected with the PLGA nanoparticle.The light microscopic image of this novel medicine carrying microvesicle is seen Fig. 3.As seen, owing to repeatedly vibrate, react, it is big that the lipid microbubble particle diameter becomes under the common light microscopic, and many medicine carrying PLGA nanoparticles are gone up in its surface connection, are petal-shaped.Fluorescence microscope down as seen, the medicine carrying PLGA nanoparticle that is connected in the lipid microbubble surface sends red fluorescence (because of the hemoporphyrin of bag in it year can spontaneous red fluorescence).
Embodiment 2
(1) preparation of common lipid microbubble:
With mass ratio 3: 1 DPPC (distearoyl phosphatidylcholine) and DSPE-PEG (2000)-NH
2, volume ratio is that 1: 4 glycerol and PBS buffer adds in the plastics tubule and mix described DPPC and DSPE-PEG (2000)-NH
2The quality summation count 3: 1 with the ratio of the volume summation of glycerol and PBS with mg/ml, 40 ℃ of heating in water bath after 40 minutes with the air in the HFC-236fa gas instead tubule, carry out making lipid microbubble behind the mechanical oscillation 50s with argental mercury capsule dispenser, the lipid microbubble particle diameter of preparation is even, good dispersion degree.
(2) preparation of medicine-carrying polymer polymer nanoparticle:
50mgPLGA (for the PLGA of band carboxyl, molecular weight 25000, polymerization ratio 50: 50) is dissolved in the 1ml dichloromethane as oil phase, adds 250 μ l sensibion aqueous solutions in oil phase, the 45s that shakes gets colostrum.With colostrum and 6ml 5%PVA (polyvinyl alcohol, molecular weight is 30000-70000) solution mixes the back with 20000r/min homogeneous dispersion 5min, get emulsion, pour 15ml 2% isopropyl alcohol again into, the room temperature lower magnetic force stirs 5h, through centrifugal, distilled water rinsing 3 times is stored in behind the lyophilization 48h in 4 ℃ of refrigerators again.Common light microscopic is observed down, and medicine-carrying polymer nanoparticle particle size distribution is even, good dispersion degree, no adhesion and gathering.Fluorescence microscope is observed down, and the hemoporphyrin that parcel is gone in the nanoparticle sends bright red fluorescence certainly.
(3) the lipid microbubble surface is charge-modified:
In the common lipid microbubble solution that step 1 makes, add 1.5ml poly-D-lysine (PBS is mixed with the solution that concentration is 1.5-2.5mg/ml), 40min is hatched in 4 ℃ of vibrations in the constant temperature oscillator, reuse PBS buffer (PH7.4) rinsing 3 times repeatedly, abandon subnatant, keep the lipid microbubble on upper strata, thereby make the positively charged lipid microbubble in surface.
(4) activated carboxylic on medicine-carrying polymer polymer nanoparticle surface
The PLGA nanoparticle of the parcel hemoporphyrin that carboxyl activator EDCHCl (1-(3-dimethyl propyl)-3-ethyl-carbodiimide hydrochloride), N-hydroxy thiosuccinimide sulfo-NHS and step 2 are made fully is mixed in the MES buffer of PH6.0 at 40: 40: 1 according to mol ratio, incubation reaction 80min under the room temperature, activation nanoparticle surface with carboxyl.Unreacted carboxyl activator is removed in the centrifugal rinsing of distilled water 3-5 time.
(5) coupling of lipid microbubble and medicine-carrying polymer polymer nanoparticle prepares the medicine carrying microvesicle
The PLGA nanoparticle that lipid microbubble that step 3 gained is positively charged and step 4 gained have an activated carboxyl fully is mixed in the PBS buffer of 0.1M of PH8.0, and 72h is hatched in vibration in 4 ℃ of constant temperature oscillators.Subnatant is abandoned in distilled water rinsing 3-5 time repeatedly, keeps the microvesicle that floats on the upper strata, promptly obtains the novel medicine carrying microvesicle that the surface is connected with the PLGA nanoparticle.As seen, owing to repeatedly vibrate, react, it is big that the lipid microbubble particle diameter becomes under common light microscopic for this novel medicine carrying microvesicle, and its surface connects goes up many medicine carrying PLGA nanoparticles, is petal-shaped.Fluorescence microscope down as seen, the medicine carrying PLGA nanoparticle that is connected in the lipid microbubble surface sends red fluorescence (because of the hemoporphyrin of bag in it year can spontaneous red fluorescence).
Embodiment 3
Save step (4) at embodiment 1 and embodiment 2, all the other are operated with embodiment 1 and 2.After centrifugal rinsing 3-5 time, the electronegative PLGA nanoparticle in activated carboxyl surface is not seen obvious the combination with surperficial positively charged lipid microbubble, the results are shown in Figure 4.Illustrate that both combinations mainly depend on chemical covalent coupling but not simple Electrostatic Absorption among embodiment 1 and the embodiment 2.
Embodiment 4
In embodiment 1 and embodiment 2, save the poly-D-lysine and the lipid microbubble reactions steps of step (3), all the other are operated with embodiment 1 and embodiment 2.After centrifugal rinsing 3-5 time, the surperficial electronegative PLGA nanoparticle of activated carboxyl is not seen obvious combination with the surface with the lipid microbubble of identical negative charge, sees Fig. 5.Explanation is in embodiment 1 and embodiment 2, and Electrostatic Absorption has also played pivotal role, and it impels two kinds of easier being in contact with one another of microgranule, has produced synergism with chemical covalent coupling.
Embodiment 5: the external release characteristics of medicine-carrying polymer polymer nanoparticle of the present invention:
The 60mg that gets the preparation of embodiment 1 and embodiment 2 steps (2) carries hemoporphyrin PLGA nanoparticle and 5mL PBS buffer (pH=7.4) is loaded in the bag filter, put into the tool plug container that fills the 50mLPBS buffer after two ends are airtight, place 37 ℃, the vibration of the shaking table constant temperature of 72r/min, respectively at the different time points 1mL that from buffer, takes a sample, in time add 1mL PBS after each sampling.Adopt reverse high performance liquid chromatography to detect,, calculate the cumulative release percentage rate (%) of different time points HP-PLGA respectively, draw the drug release time curve according to its concentration of standard curve Equation for Calculating and cumulative release amount.External drug release time curve is seen Fig. 6.As can be seen from Fig. 6,24h HP-PLGA drug release rate is that 36.5%, the 2d-12d rate of release becomes gently, and rate of release is accelerated after the 12d, to 14d altogether release reach 88.3%.The medicine-carrying polymer polymer nanoparticle release that the method for the invention preparation is described is slow, can be used as the sustained-release and controlled release carrier of medicine.
Embodiment 6: the experiment of developing in the body of medicine carrying microvesicle of the present invention
Behind the 4% pentobarbital sodium 30mg/kg muscle anesthetized rabbit, dorsal position is fixed, depilation.Use Philips iU22 colorful Doppler ultrasound diagnostic apparatus (L12-5 probe, frequency probe 7-13MHz), adopt self cross-reference method, conventional first-harmonic scanning obtains the Hepar Leporis seu Oryctolagi ultrasonogram.During radiography imaging modality is adjusted to second harmonic, the novel medicine carrying microvesicle of embodiment 1 and embodiment 2 preparations after the 0.5ml dilution is annotated by auricular vein group, time of developing and imaging results behind the observation liver radiography.Visual observations under the harmonic wave state behind the microbubble 3S of injection embodiment 1 preparation, the contrast agent filling occurs in the liver blood vessel, the appearance of liver parenchyma echo strengthens behind the 5S, and liver parenchyma echo obviously strengthens after a few minutes, still as seen strengthens behind the 20min.The medicine carrying microvesicle that the method for the invention preparation is described can be used as acoustic contrast agent.
It below only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (11)
1. the preparation method of a medicine carrying microvesicle may further comprise the steps:
Step 1: distearoyl phosphatidylcholine, another kind of phospholipid, glycerol are mixed with phosphate buffer and heat, the mass ratio of described distearoyl phosphatidylcholine and another kind of phospholipid is 2-3: 1, the volume ratio of glycerol and PBS is 1: 2-4, and the quality summation of DPPC and another kind of phospholipid is counted 3-6 with the ratio of the volume summation of glycerol and PBS with mg/ml: 1; Charge into perfluoropropane gas or HFC-236fa gas vibration 40-50s, standby;
Step 2: be dissolved in the 1ml dichloromethane containing the macromolecule polymeric material 40-50mg that holds carboxyl in the molecule side group, add 200-250 μ l medicine, the sound 35-45s that shakes gets colostrum; Colostrum is mixed with the 3-6ml3%-5% poly-vinyl alcohol solution, with 20000-23000r/min homogeneous dispersion effect 5min, add 8-15ml 2%-4% isopropyl alcohol again, stir 2-5h, it is standby to be the medicine-carrying polymer polymer nanoparticle after rinsing, lyophilization;
Step 3: add positively charged biochemical reagents in step 1 gained solution, the constant temperature vibration is hatched, and the lipid microbubble of leaving and taking the upper strata is standby;
Step 4: it is that 0.1M, pH are in 6.0 the buffer solution that the medicine-carrying polymer polymer nanoparticle that N-hydroxy thiosuccinimide and another kind of carboxyl activator and step 2 are made fully is mixed in concentration, incubation reaction 30-80min under the room temperature, centrifugal rinsing, get the precipitation standby;
Step 5: it is that 0.1M, pH are in 8.0 the buffer solution that the lipid microbubble of step 3 gained and step 4 gained precipitation fully are mixed in concentration, 0 ℃-4 ℃ hatch 25-40min with 30-45r/min vibration after, leave standstill at 0 ℃-4 ℃ and to hatch 24-72h, leave and take the microvesicle on upper strata.
2. preparation method according to claim 1 is characterized in that, the described phospholipid of step 1 is DSPE-PEG (2000)-NH
2, described being heated to be at 35 ℃-40 ℃ heating 30-40min.
3. preparation method according to claim 1 is characterized in that, the described macromolecule polymeric material of step 2 is a kind of in polylactic acid, the polylactic acid-glycolic guanidine-acetic acid copolymer.
4. preparation method according to claim 1 is characterized in that, the described positively charged biochemical reagents of step 3 are poly-l-lysine, and molecular weight is 3-7 ten thousand, and addition is 1.5-3.5mg.
5. preparation method according to claim 1 is characterized in that, step 30 ℃-4 ℃ of temperature with 30-45r/min vibration 25-40min.
6. preparation method according to claim 1 is characterized in that, carboxyl activator described in the step 4 is 1-(3-dimethyl propyl)-3-ethyl carbodiimide or 1-(3-dimethyl propyl)-3-ethyl-carbodiimide hydrochloride.
7. preparation method according to claim 1 is characterized in that, the mol ratio of the hydroxy thiosuccinimide of N-described in the step 4, another kind of carboxyl activator and medicine-carrying polymer polymer nanoparticle is all greater than 30: 1.
8. preparation method according to claim 1 is characterized in that, the described centrifugal speed of step 4 is 3000-3500r/min, centrifugal 5min.
9. preparation method according to claim 1 is characterized in that, buffer described in step 4 and the step 5 is a kind of in PBS buffer, MES buffer, borate buffer solution, the Tris buffer.
10. according to the medicine carrying microvesicle of each described preparation method preparation of claim 1-9, it is characterized in that, described medicine carrying microvesicle is made up of lipid microbubble and medicine-carrying polymer polymer nanoparticle, and described medicine-carrying polymer polymer nanoparticle is connected with lipid microbubble by amido link.
11. medicine carrying microvesicle according to claim 10 is characterized in that, the particle diameter of described medicine-carrying polymer polymer nanoparticle is 550-650nm.
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| CN104096245A (en) * | 2014-07-18 | 2014-10-15 | 重庆医科大学 | Ultrasound lipid microbubble wrapping drug-carrying albumin nanoparticles and preparation method thereof |
| CN104755108A (en) * | 2012-10-25 | 2015-07-01 | 松岗大学研究基金会 | Ultrasound contrast medium in which nanoparticles containing drug are combined, and preparation method therefor |
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