CN102525935A - Brain delivery method for nano-medicament carrier - Google Patents

Brain delivery method for nano-medicament carrier Download PDF

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Publication number
CN102525935A
CN102525935A CN2012100149356A CN201210014935A CN102525935A CN 102525935 A CN102525935 A CN 102525935A CN 2012100149356 A CN2012100149356 A CN 2012100149356A CN 201210014935 A CN201210014935 A CN 201210014935A CN 102525935 A CN102525935 A CN 102525935A
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nano
medicament carrier
ultrasonic
microvesicle
meant
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CN2012100149356A
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Chinese (zh)
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高小玲
姚磊
陈红专
宋清香
胡兵
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上海交通大学医学院
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Priority to CN2012100149356A priority Critical patent/CN102525935A/en
Publication of CN102525935A publication Critical patent/CN102525935A/en

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Abstract

The invention provides a brain delivery method for a nano-medicament carrier. Brain delivery of the nano-medicament carrier carrying a diagnosis and treatment medicament is mediated by using ultrasonic waves and micro-vesicles together; the nano-medicament carrier and the micro-vesicles are administrated separately or administrated simultaneously after being mixed easily or administrated simultaneously through non-covalent and covalent connection; and in the brain delivery of the nano-medicament carrier carrying the diagnosis and treatment medicament under the mediation of ultrasonic waves and micro-vesicles, ultrasonic treatment is performed immediately or a certain time interval after micro-vesicles are applied to a mechanism. Compared with the conventional brain delivery of a medicament under the mediation of ultrasonic waves and micro-vesicles, the method has the advantages that: poor in-vivo properties (poor stability, easiness in clearing by the organism and the like) of the medicament can be eliminated, the diagnosis molecular signal strength is enhanced, the detection sensitivity is enhanced, and focus targeting can be realized; and compared with the conventional brain delivery technology for the nano-medicament carrier, the method has the advantage that: external ultrasonic waves are applied in a technology, so that more efficient and more specific brain medicament delivery can be realized.

Description

Delivering method in a kind of nano-medicament carrier brain

Technical field

The present invention relates to the medicine and pharmacology field, more particularly, relate to delivery technique in the microbubble mediated nano-medicament carrier brain of a kind of ultrasonic in combination.

Background technology

Brain diseases comprises the cerebral tumor and neurodegenerative diseases (like alzheimer disease, parkinson disease) etc., is the major disease of harm humans life and health, and sickness rate just is being the trend that rises year by year.Yet the active drug diagnosis and treatment of brain diseases at present still face several big core difficult problems, and (Blood Brain Barrier, BBB) administration is a primary difficult problem that realizes that the brain diseases Drug therapy must overcome wherein to cross over blood brain barrier.BBB is one deck barrier system that is present between blood and the cerebral tissue; Closely connect and compose by the iuntercellular of polar brain capillary endothelial cell through complicacy; It is important to maintaining in the brain the relatively stable ten minutes of environment, but also is simultaneously the main barrier that medicine gets into cerebral tissue performance prevention, treatment and diagnostic effect.After the routine clinical preparation administration, 98% chemicals and be close to 100% albumen/polypeptide and genomic medicine and all be difficult to greatly influence treatment effect to brain diseases into brain.Therefore, the design construction medicine delivery technique of efficiently crossing over BBB has epochmaking researching value and application prospect.

Sent for increasing in the medicine brain clinically in the past, often adopted the open blood brain barrier of perfusion of carotid artery hyperosmotic solution, or through sphenotresia, medicine injection in the local brain.These methods exist brain injury, hemorrhage and infection equivalent risk.Improving the research of sending in the medicine brain with the Non-Invasive route of administration is research emphasis and the focus of domestic in recent years external-brain's internal target to administration.Wherein the brain targeted nano delivery system of receptor, absorption mediation receives great concern because of having excellent drug carrier band and delivery capability.Yet these class methods still exist, and tissue selectivity is relatively poor, brain drug delivery efficient is not ideal enough, be difficult to accurate targeting drug delivery to problems such as focuses.Main cause is: the receptor that 1. relates to does not often exist only in the brain blood capillary, go back wide expression in other peripheral organs, so tissue selectivity is relatively poor; 2. receive the restriction of factors such as expression of receptor amount, affinity, transport efficacy and endogenous material influence, such preparation is compared with non-targeting preparation, and brain drug delivery efficient improves limited, is difficult to realize from the basis to clinical conversion.

Along with the development of modern acoustics technology, ultrasonic technique has been widely used in routine diagnosis, also just progressively is used to the treatment field, like the tumor enhanced sensitivity, kill and wound and strengthen the biomembrane permeability, promotes transport of drug, gene transfered cell etc.In recent years, ultrasonic means as the of short duration BBB of opening of a kind of physical property brain that is used to improve medicine is sent and is received increasing concern.Nineteen fifty-five, Barnard etc. report that the earliest medical supersonic can improve the BBB permeability, but the simple ultrasonic BBB of opening that discovers afterwards needs higher energy, when improving the BBB permeability, often causes cerebrovascular or brain tissue impairment.Until calendar year 2001, using ultrasounds such as Hynynen associating microvesicle technology just makes this problem be able to solve.Microvesicle is a kind of micron order bubble of the inside air inclusion that is prepared from the good albumin of biocompatibility, saccharide, nonionic surfactant, lipid or Biodegradable polymer material; Its acting body under ultrasonic field is present: 1. as powerful acoustic reflection body and scattering object; Microvesicle can strengthen the echo ability of tissue greatly; The ultrasonoscopy of fine definition and contrast can be provided under the ultrasound condition of gentleness, be beneficial to diagnosis; 2. under ultrasonication; Microvesicle produces cavitation effect (cavatition) because of vibration; But induction of vascular endothelial cell traffic vesicle (vacuole) forms; Increase the endocytosis transhipment, and the closely of short duration opening of connection of mediation parts of fine intercellular, selectivity improves the permeability of ultrasonication position tunica vasculose thus.Under the microvesicle existence condition, therefore ultrasonic raising BBB permeability power demand greatly reduces the brain tissue injury risk than more than simple ultrasonic reduction by two one magnitude.With other brain targeting drug delivery compared with techniques, the microbubble mediated medicine of ultrasonic in combination strides that BBB transhipment has that tissue selectivity is strong, efficient, noinvasive and advantage such as reversible.Therefore, the microbubble mediated brain drug delivery technology of ultrasonic in combination is a kind of very promising novel brain targeting drug delivery technology.Blemish in an otherwise perfect thing be the medicine that itself has undesirable property such as poor stability, strongly hydrophilic for some, its efficient of going into brain of ultrasonic in combination microvesicle technology mediation is still not ideal enough.In addition, this technology lacks targeting characteristic biologically, still can't further realize molecular targeted to the intracerebral lesion position.

Have efficient medicine carrying ability and can remedy the technological above-mentioned deficiency of ultrasonic in combination microvesicle largely with the nano-medicament carrier (like nanoparticle, liposome, vesicle, micelle etc.) that can realize molecular targeted function, and the complementation good with its formation.Nano-medicament carrier has following characteristic: 1. through bag medicine carrying thing, shelter the physicochemical property that itself is unfavorable for seeing through blood brain barrier, efflux characteristic etc. like hydrophilic and P-glycoprotein, performance nanoscale effect imports in the brain of increase medicine; 2. protect medicine to avoid the degraded of enzyme and environment pH, improve the particularly bioavailability of biopharmaceutical macromolecular drug of medicine; 3. bag carries diagnostic medicine, but enhancing signal intensity improves detection sensitivity, improves imaging effect; 4. can carry out the target function molecular modification to nano-medicament carrier according to Biological Principles, utilize its initiatively targeting function, increase the affinity of nano-medicament carrier, realize medicine concentrating at lesions position to focus.

Based on this, the present invention proposes a kind of novel brain targeting drug delivery strategy: combine the modern acoustics technology to pass the medicine technology with nanometer, send (Fig. 1) in the efficient brain of the nano-medicament carrier through ultrasonic in combination microvesicle technology mediation year diagnosis and treatment medicine.

Summary of the invention

Technical problem to be solved by this invention is that a kind of interior delivering method of efficient brain that utilizes the nano-medicament carrier of microbubble mediated year diagnosis and treatment medicine of ultrasonic in combination is provided.

Second technical problem to be solved by this invention is that a kind of Nano medication compositions is provided.

In order to solve above-mentioned first problem; The invention provides delivering method in a kind of nano-medicament carrier brain; Said delivering method is meant in the brain of the nano-medicament carrier that utilizes microbubble mediated year diagnosis and treatment medicine of ultrasonic in combination and sends; Said nano-medicament carrier and microvesicle for administration respectively, simply mix administration simultaneously, non-covalently be connected a kind of in administration simultaneously or the covalently bound while administration, send model of action in the brain of the nano-medicament carrier of microbubble mediated year diagnosis and treatment medicine of said ultrasonic in combination and be body and give behind the microvesicle ultrasonic immediately or ultrasonic after a while.

Said administration respectively is meant nano-medicament carrier administration in 4 hours before or after ultrasonic microbubble is handled; Preferably administration in 2 hours after ultrasonic microbubble is handled.

Said simple mixing administration simultaneously is meant that preceding microvesicle of administration and nano-medicament carrier simply mix (not having special interaction), administration simultaneously then.

Said non-covalent connection administration simultaneously is meant that nano-medicament carrier and microvesicle are through the non-covalent combination of electrostatic interaction; Said covalently bound while administration is meant through surface-functionalized group of nano-medicament carrier and microvesicle covalently bound.

Saidly be meant that through the non-covalent combination of electrostatic interaction using positively charged phospholipid or derivatives thereof prepares microvesicle, the nano-medicament carrier that absorption surface is electronegative; Saidly be meant that through the surface-functionalized group of nano-medicament carrier and microvesicle are covalently bound to prepare microvesicle through the phospholipid derivative of using band functional group such as SPDP covalently bound with the nano-medicament carrier of surface band amino functional group, perhaps using the amino phospholipid derivative of band functional group such as maleimide, to prepare microvesicle covalently bound with the nano-medicament carrier of surface band sulfydryl functional group.

As one preferred, said body give behind the microvesicle ultrasonic after a while be meant give behind the microvesicle ultrasonic in 10 minutes.

The said ultrasonic pressure wave that is meant that high frequency changes, ultrasonic frequency range is 20 KHz-10 MHz, preferred 0.2MHz-5 MHz; The mechanical index scope is 0.1-2.0, preferred 0.6-1.4.

Said microvesicle is meant that with albumin, phospholipid, phospholipid derivant or high molecular polymer be material; With perfluoropropane, perfluorinated butane or sulfur hexafluoride serves as to fill gas, gets the micron order bubble with cross-linking method, probe ultrasonic method, freeze-drying, emulsion process, mechanical oscillation method, coaxial unidirectional current atomization, flow focusing method or the preparation of micro-fluidic method; Said albumin is meant one or more in human serum albumin or the bovine serum albumin; Said phospholipid, phospholipid derivant are meant one or more in sphingomyelins, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine, phosphatidyl glycerol, phosphatidylinositols, phosphatidic acid and the derivant thereof, and said high molecular polymer is meant one or more in polylactic acid (PLA) or the poly lactic coglycolic acid (PLGA); Said microvesicle particle size range is 0.1-20 μ m, is preferably 0.5-10 μ m.

The particle size range of said nano-medicament carrier is 1-1000 nm, is preferably 10-200 nm.

In order to solve second problem of the present invention; The invention provides a kind of Nano medication compositions; Contain nano-medicament carrier and medicine; Said Nano medication compositions also contains microvesicle; Said microvesicle is meant that with albumin, phospholipid, phospholipid derivant or high molecular polymer be material, serves as to fill gas with perfluoropropane, perfluorinated butane or sulfur hexafluoride, gets the micron order bubble with cross-linking method, probe ultrasonic method, freeze-drying, emulsion process, mechanical oscillation method, coaxial unidirectional current atomization, flow focusing method or the preparation of micro-fluidic method; Said nano-medicament carrier is meant according to what conventional method prepared can wrap the nanoscale complex that carries and carry medicine; Said medicine is meant maincenter disease treatment and diagnostic medicine; Or pass through the medicine that central action is brought into play the systemic treatment effect; Said drug encapsulation is in nano-medicament carrier; Be in micromolecule chemicals, diagnostic medicine, polypeptide protein medicine and the genomic medicine one or several, said Nano medication compositions is according to delivering method targeting drug delivery in the said brain.

Delivering method is meant in the brain of the nano-medicament carrier that utilizes microbubble mediated year diagnosis and treatment medicine of ultrasonic in combination and sends in the said brain; Said nano-medicament carrier and microvesicle for administration respectively, simply mix administration simultaneously, non-covalently be connected a kind of in administration simultaneously, the covalently bound while administration, send model of action in the brain of the nano-medicament carrier of microbubble mediated year diagnosis and treatment medicine of said ultrasonic in combination and be body and give behind the microvesicle ultrasonic immediately or ultrasonic after a while.

Said administration respectively is meant nano-medicament carrier administration in 4 hours before or after ultrasonic microbubble is handled; Preferably administration in 2 hours after ultrasonic microbubble is handled.

Said simple mixing administration simultaneously is meant that preceding microvesicle of administration and nano-medicament carrier simply mix (not having special interaction), administration simultaneously then.

Said non-covalent connection administration simultaneously is meant that nano-medicament carrier and microvesicle are through the non-covalent combination of electrostatic interaction; Said covalently bound while administration is meant through surface-functionalized group of nano-medicament carrier and microvesicle covalently bound.

Saidly be meant that through the non-covalent combination of electrostatic interaction using positively charged phospholipid or derivatives thereof prepares microvesicle, the nano-medicament carrier that absorption surface is electronegative; Saidly be meant that through the surface-functionalized group of nano-medicament carrier and microvesicle are covalently bound to prepare microvesicle through the phospholipid derivative of using band functional group such as SPDP covalently bound with the nano-medicament carrier of surface band amino functional group, perhaps using the amino phospholipid derivative of band functional group such as maleimide, to prepare microvesicle covalently bound with the nano-medicament carrier of surface band sulfydryl functional group.

As one preferred, said body give behind the microvesicle ultrasonic after a while be meant give behind the microvesicle ultrasonic in 10 minutes.

The said ultrasonic pressure wave that is meant that high frequency changes, ultrasonic frequency range is 20 KHz-10 MHz, preferred 0.2MHz-5 MHz; The mechanical index scope is 0.1-2.0, preferred 0.6-1.4.

Nano-medicament carrier of the present invention is meant according to what conventional method prepared can wrap the nanoscale complex that carries and carry medicine, is meant that specifically nanoparticle with the preparation of pharmaceutics means, vesicle, liposome, micelle are or/and the complex of diagnosis and treatment medicine.

Described nanoparticle is selected macromolecular material polyesters Biodegradable material and derivant preparation thereof for use.Preferred polyethylene glycol-lactic acid block copolymer (PEG-PLA), Polyethylene Glycol-lactic-co-glycolic acid block copolymer (PEG-PLGA) and polyethylene glycol-caprolactone (PEG-PCL).Wherein polyalkylene glycol moiety is mono methoxy polyethylene glycol or functional polyalkylene glycol.Functional groups is a kind of in dimaleoyl imino, sulfydryl, hydrazide group, carboxyl, azido, aldehyde radical, biotin or the Avidin.The nanoparticle surface can connect the part of targeting intracerebral lesion through the functionalization group, like the D type polypeptide of Beta amyloid plaques in the RGD peptide of targeting cerebral tumor blood vessel, the targeting brain etc.

Described vesicle material selection polyethylene glycol-lactic acid block copolymer (PEG-PLA) is or/and functional PEG-PLA; Polyethylene Glycol-lactic-co-glycolic acid block copolymer (PEG-PLGA) is or/and functional PEG-PLGA; Polyethylene glycol-caprolactone block copolymer (PEG-PCL) is or/and functional PEG-PCL.Wherein, the functional groups of functional polyalkylene glycol part is a kind of in dimaleoyl imino, sulfydryl, hydrazide group, carboxyl, azido, aldehyde radical, biotin or the Avidin.The vesicle surface can connect the part of targeting intracerebral lesion through the functionalization group, like the D type polypeptide of Beta amyloid plaques in the RGD peptide of targeting cerebral tumor blood vessel, the targeting brain etc.

In described liposome material selection sphingomyelins, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine, phosphatidyl glycerol, phosphatidylinositols, phosphatidic acid or cholesterol and the derivant thereof one or more.Surface of liposome can connect the part of targeting intracerebral lesion through the functionalization group, like the D type polypeptide of Beta amyloid plaques in the RGD peptide of targeting cerebral tumor blood vessel, the targeting brain etc.

Described micelle is selected the amphipathic nature polyalcohol material preparation for use.Preferred polyethyleneglycol block copolymer is like polyethylene glycol-lactic acid block copolymer (PEG-PLA), polyethylene glycol-lactic acid lactic-co-glycolic acid block copolymer (PEG-PLGA) and polyethylene glycol-caprolactone (PEG-PCL), poloxamer etc.Wherein polyalkylene glycol moiety is mono methoxy polyethylene glycol or functional polyalkylene glycol.Functional groups is a kind of in dimaleoyl imino, sulfydryl, hydrazide group, carboxyl, azido, aldehyde radical, biotin or the Avidin.Micellar surface can connect the part of targeting intracerebral lesion through the functionalization group, like the D type polypeptide of Beta amyloid plaques in the RGD peptide of targeting cerebral tumor blood vessel, the targeting brain etc.

It is in micromolecule chemicals, diagnostic medicine, polypeptide protein medicine and the genomic medicine one or several that bag is stated from medicine in the nano-medicament carrier; Mainly be maincenter disease treatment and diagnostic medicine, or bring into play the medicine of systemic treatment effect through central action.Wherein diagnostic medicine is nuclear magnetic resonance agent or radio nuclide imaging agent, preferred gadolinium agent and Fe 3O 4

The administering mode of microvesicle of the present invention and nano-medicament carrier is intravenous injection or intramuscular injection.

The invention has the advantages that; Compare with sending in the microbubble mediated medicine brain of traditional ultrasonic in combination, this technology can overcome character in the bad body of medicine (poor stability, be prone to by the body removing etc.), strengthens diagnosis molecular signal intensity; Improve detection sensitivity, can realize the focus targeting; And with existing nano-medicament carrier brain drug delivery compared with techniques, this technology ultrasonicly realizes more efficiently through adding, medicine is sent in the more special brain.

Description of drawings

Fig. 1 sends sketch map in the brain of the microbubble mediated nano-medicament carrier of ultrasonic in combination.

Fig. 2 is the microbubble mediated nanoparticle brain drug delivery of ultrasonic in combination, curve chart when cortex, Hippocampus, basal nuclei and blood plasma medicine, and with the independent drug administration by injection matched group of nanoparticle relatively.

Fig. 3 is that ultrasonic in combination coumarin 6 liposome transhipment in microbubble mediated year gets into brain essence.A, the independent drug administration by injection of liposome; Liposome drug administration by injection immediately after B, ultrasonic in combination microvesicle handle.1, the liposome signal; 2, liposome signal+blood vessel signal; 3, liposome signal+blood vessel signal+nucleus signal.

Fig. 4 is the microbubble mediated Fe of ultrasonic in combination 3O 4The cerebrovascular endothelial cell of nanoparticle and neuronal cell picked-up, wherein * refers to Fe 3O 4Nanoparticle, short arrow is represented the endocytosis vesicle, and long arrow representative closely connects.

The specific embodiment

Below in conjunction with accompanying drawing the specific embodiment provided by the invention is elaborated.

Send in the brain of embodiment 1, microbubble mediated year coumarin 6 nanoparticle of ultrasonic in combination

Carry the preparation of coumarin 6 nanoparticle: mono methoxy polyethylene glycol-polylactic acid (MPEG-PLA) block copolymer 10-25 mg is dissolved in the 1 ml dichloromethane solution (containing 0.01-0.2 mg coumarin 6); The sodium cholate solution that adds 2 ml 1%, 220 W continuous ultrasounds, 30 s-2 min obtain colostrum.Colostrum joins in the sodium cholate solution of 8-25 ml 0.5%; Rotary evaporation is removed dichloromethane after stirring 5 min; 4 ℃ of 14000 centrifugal 45 min of rpm removes supernatant, disperses again with 0.01 mol/L pH7.0 HEPES; Cross Sepharose CL-4B gel column and remove and do not seal coumarin 6, promptly get and carry the coumarin 6 nanoparticle.

Adopt clinical diagnosis to use Vltrasonic device, the SonoVue (the about 2 μ m of particle diameter) that uses in China with the SFDA approval is ultrasonic microbubble.Investigate ultrasound mode, mechanical index, time, microvesicle dosage, nanoparticle delivery time to carrying the influence of sending in the coumarin 6 nanoparticle brain like table 1.The result shows, the ultrasonic in combination microvesicle gives nanoparticle after handling immediately, and it is best to go into the brain effect; Type B radiography pattern promotes that sending effect in the nanoparticle brain is superior to the quickflashing pattern; Microvesicle dosage is gone into the brain effect to nanoparticle has certain influence, and 30 a μ l/ effect only are superior to 60 μ l/; Mechanical index is to influence the key factor of sending effect in the nanoparticle brain: mechanical index is 0.6, and nanoparticle is gone into brain volume and nanoparticle, and to be injected into brain volume separately similar; Mechanical index 0.9, nanoparticle are gone into the twice that brain volume is a matched group; Mechanical index is increased to 1.1,1.4, and nanoparticle goes into brain volume and mechanical index is 0.9 group of there was no significant difference.

Table 1. ultrasonic in combination microvesicle Processing Test parameter (supersonic frequency is 0.2-5 MHz).

? Microvesicle dosage (μ l) Ultrasound mode Mechanical index Ultrasonic time Nanoparticle is injected opportunity 1 - - - - ? 2 30 - - - Injection immediately after ultrasonic microbubble is handled 3 - B 0.9 1 min Injection immediately after ultrasonic microbubble is handled 4 30 B 0.9 1 min Injection immediately after ultrasonic microbubble is handled 5 30 B 0.9 1 min Ultrasonic microbubble is handled back 2 h injection 6 30 B 0.9 1 min Ultrasonic microbubble is handled back 4 h injection 7 30 The quickflashing pattern 1.2 1 min Injection immediately after ultrasonic microbubble is handled 8 60 B 0.9 1 min Injection immediately after ultrasonic microbubble is handled 9 30 B 0.9 20 s*3 time Injection immediately after ultrasonic microbubble is handled 10 30 B 0.6 1 min Injection immediately after ultrasonic microbubble is handled 11 30 B 1.1 1 min Injection immediately after ultrasonic microbubble is handled 12 30 B 1.4 1 min Injection immediately after ultrasonic microbubble is handled

Supersonic frequency is 2.5 MHz, and mechanical index 0.9, continuous ultrasound 1 min, microvesicle dosage 30 μ l/ are only injected immediately after ultrasonic microbubble is handled and carried the coumarin 6 nanoparticle.Nanoparticle injection back different time points HPLC-FD detects coumarin 6 concentration in cerebral tissue and the blood plasma.The result is as shown in Figure 2, and the ultrasonic in combination microvesicle significantly strengthens (cortex, Hippocampus and basal nuclei) coumarin 6 abundance in the brain, does not distribute and do not change its blood circulation, but shows that ultrasonic in combination microvesicle site specific strengthens the nanoparticle transhipment in the brain.

Embodiment 2, ultrasonic in combination coumarin 6 liposome transhipment in microbubble mediated year get into brain essence

Carry the preparation of coumarin 6 liposome: 5-30 mg DMPC, 0.01-1 mg coumarin 6 are dissolved in the 2 ml chloroforms, and rotary evaporation obtains adipose membrane, add 10 ml solution and carry out aquation, obtain multilamelar liposome.200 W probe is ultrasonic, high pressure homogenize or crowded embrane method make small unilamellar vesicle, crosses gel column and separates to remove and does not seal coumarin 6.

Adopt clinical diagnosis to use Vltrasonic device, SonoVue is a ultrasonic microbubble.The ultrasonic in combination microvesicle gives liposome after handling immediately, and experiment mice normal saline cardiac perfusion is removed blood behind 3 h, and 4% paraformaldehyde is fixed.Get brain, 4% paraformaldehyde continues fixing 24 h, and 10%, 20% sucrose solution dewaters successively, OCT embedding, frozen section.20% lowlenthal serum of cutting into slices seals 1 h, Alexa Fluor The anti-Mus CD31 of 647 labellings antibody (1:100) 4 oThe C incubated overnight, PBS washes 3 times, and DAPI dyes 8 min, and Zeiss LSM 710 Laser Scanning Confocal Microscopes are observed.

The result shows (Fig. 3), and a large amount of fluorescent labeling liposomees of ultrasonic in combination microvesicle group are transported through cerebrovascular, gets into brain essence; And liposome is injected matched group brain lactone plastid abundance separately far fewer than the ultrasonic microbubble processed group.

Embodiment 3, the microbubble mediated mediation of ultrasonic in combination Fe 3O 4The cerebrovascular endothelial cell of nanoparticle and neuronal cell picked-up

Adopt clinical diagnosis to use Vltrasonic device, SonoVue is a ultrasonic microbubble.The ultrasonic in combination microvesicle gives Fe after handling immediately 3O 4Nanoparticle, experiment mice normal saline cardiac perfusion is removed blood behind 3 h, and 2.0% glutaraldehyde is fixed.Get brain, cut Hippocampus and locate 1 cm 3Piece of tissue, 2% glutaraldehyde continue fixing 24 h, and osmic acid is fixed, embedding, ultrathin section, the situation that microbubble mediated BBB junction opened of transmission electron microscope observing ultrasonic in combination and transitional vesicle form.

The result shows (Fig. 4), and tangible endocytosis vesicle, Fe appear in ultrasonic in combination microvesicle group cerebrovascular endothelial cell 3O 4Nanoparticle is absorbed by brain endothelial cell, and part gets into neuronal cell; And independent Fe 3O 4Tangible endocytosis vesicle does not appear in nanoparticle intravenous injection group, does not see cerebrovascular endothelial cell picked-up Fe 3O 4Nanoparticle.

Beta amyloid plaques position in embodiment 4, the peptide modified nanoparticle targeting of the microbubble mediated D type of the ultrasonic in combination brain

A certain proportion of methoxy poly (ethylene glycol)-lactic-co-glycolic acid block copolymer (MPEG-PLGA) and dimaleoyl imino Polyethylene Glycol-lactic-co-glycolic acid block copolymer (Maleimide-PEG-PLGA) are made nanoparticle (NP) through emulsifying/solvent evaporation.Entrust the synthetic D type polypeptide D-pep of polypeptide Synesis Company, and at its terminal cysteine that connects.Under condition of neutral pH, the maleimide base group and the D-pep on above-mentioned nanoparticle surface go up the sulfydryl covalent bond that contains in the cysteine, and centrifugalize is removed and do not connected polypeptide, obtains D-pep-NP.

Adopt clinical diagnosis to use Vltrasonic device, SonoVue is a ultrasonic microbubble.The ultrasonic in combination microvesicle gives D-pep-NP after handling immediately.AD experimental animal model quick aging mice SAMP8 normal saline cardiac perfusion is removed blood behind 3 h, and 4% paraformaldehyde is fixed.Get brain, 4% paraformaldehyde continues fixing 24 h, and 10%, 20% sucrose solution dewaters successively, OCT embedding, frozen section.20% lowlenthal serum of cutting into slices seals 1 h, and trypan blue dyes beta amyloid plaques in the brain, and PBS washes 3 times, and DAPI dyes 8 min, and Zeiss LSM 710 Laser Scanning Confocal Microscopes are observed.

The result shows; The D-pep-NP and the trypan blue that carry fluorescent probe coumarin-6 dye the special site specific that exists of amyloid plaques deposition site and locate altogether; Transhipment in the microbubble mediated nanoparticle brain of prompting ultrasonic in combination; D-pep-NP can further assemble to beta amyloid plaques deposition site after going into brain, realizes the intracerebral lesion targeting.

The covalently bound while administration of embodiment 5, microvesicle and nanoparticle is sent in the efficient brain of ultrasonic mediation nanoparticle

DPPC, DSPE-PEG 2000-maleimide feeds intake with the 9:1 molar ratio, puts round-bottomed flask, adds CHCl 3Dissolving, rotary evaporation is removed organic solvent.The lipid film that forms disperses with glycerol, propylene glycol, water (1:2:7) mixed solvent.The lipid soln that obtains shifts to divide and is filled to 2.5 ml cillin bottles, and C is filled on cillin bottle top 4F 10Gas, the bottleneck sealing, high speed machine vibration (4000 rpm, 15 s) promptly gets the functionalization microvesicle.

Mono methoxy polyethylene glycol-polylactic acid (MPEG-PLA), sulfydryl-polyethylene glycol-lactic acid (HS-PEG-PLA) block copolymer feed intake with the 9:1 molar ratio; Add the dichloromethane solution dissolving that 1 ml contains a certain amount of near infrared fluorescent probe DIR; Add in the sodium cholate solution of 2 ml 1%; 220 W are interrupted (1 s/ time) ultrasonic 30 times, obtain the o/w breast.Add in the sodium cholate solution of 25 ml 0.5%, stir 5 min after rotary evaporation remove dichloromethane, 4 ℃ of 14000 centrifugal 45 min of rpm removes supernatant, disperses promptly to get the functionalized nano grain again with 0.01 mol/L pH7.0 HEPES.

Functionalization microvesicle and functionalized nano grain are pressed maleimide and sulfydryl 1:1 or 3:1 molar ratio hybrid reaction 4-8 h, promptly get and carry the nanoparticle microvesicle.Intravenous injection, ultrasonic immediately, the toy living imaging is observed behind 3 h.The result shows, it is individually dosed that ultrasonic group of brain fluorescence intensity far is better than with the dosage nanoparticle, shows to go into brain volume by the covalently bound nanoparticle of ultrasonic mediation and microvesicle significantly to be superior to nanoparticle individually dosed.

The above only is a 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 improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.

Claims (11)

1. delivering method in the nano-medicament carrier brain; It is characterized in that; Said delivering method is meant in the brain of the nano-medicament carrier that utilizes microbubble mediated year diagnosis and treatment medicine of ultrasonic in combination and sends; Said nano-medicament carrier and microvesicle for administration respectively, simply mix administration simultaneously, non-covalently be connected a kind of in administration simultaneously or the covalently bound while administration, send model of action in the brain of the nano-medicament carrier of microbubble mediated year diagnosis and treatment medicine of said ultrasonic in combination and be body and give behind the microvesicle ultrasonic immediately or ultrasonic after a while.
2. delivering method in the nano-medicament carrier brain according to claim 1 is characterized in that, said administration respectively is meant nano-medicament carrier administration in 4 hours before or after ultrasonic microbubble is handled.
3. delivering method in the nano-medicament carrier brain according to claim 2 is characterized in that, said administration respectively is meant nano-medicament carrier administration in 2 hours after ultrasonic microbubble is handled.
4. delivering method in the nano-medicament carrier brain according to claim 1 is characterized in that, said simple mixings administration simultaneously is meant that microvesicle and nano-medicament carrier simply mix while administration then before administration.
5. delivering method in the nano-medicament carrier brain according to claim 1 is characterized in that, said non-covalent connection administration simultaneously is meant that nano-medicament carrier and microvesicle are through the non-covalent combination of electrostatic interaction; Said covalently bound while administration is meant through surface-functionalized group of nano-medicament carrier and microvesicle covalently bound.
6. delivering method in the nano-medicament carrier brain according to claim 5; It is characterized in that; Saidly be meant that through the non-covalent combination of electrostatic interaction using positively charged phospholipid or derivatives thereof prepares microvesicle, the nano-medicament carrier that absorption surface is electronegative; Saidly be meant that through the surface-functionalized group of nano-medicament carrier and microvesicle are covalently bound to prepare microvesicle through the phospholipid derivative of using band functional group such as SPDP covalently bound with the nano-medicament carrier of surface band amino functional group, perhaps using the amino phospholipid derivative of band functional group such as maleimide, to prepare microvesicle covalently bound with the nano-medicament carrier of surface band sulfydryl functional group.
7. delivering method in the nano-medicament carrier brain according to claim 1 is characterized in that, said body give behind the microvesicle ultrasonic after a while be meant give behind the microvesicle ultrasonic in 10 minutes.
8. delivering method in the nano-medicament carrier brain according to claim 1 is characterized in that, the said ultrasonic pressure wave that is meant that high frequency changes, and ultrasonic frequency range is 20 KHz-10 MHz, preferred 0.2MHz-5 MHz; The mechanical index scope is 0.1-2.0, preferred 0.6-1.4.
9. delivering method in the nano-medicament carrier brain according to claim 1; It is characterized in that; Said microvesicle is meant that with albumin, phospholipid, phospholipid derivant or high molecular polymer be material; With perfluoropropane, perfluorinated butane or sulfur hexafluoride serves as to fill gas, gets the micron order bubble with cross-linking method, probe ultrasonic method, freeze-drying, emulsion process, mechanical oscillation method, coaxial unidirectional current atomization, flow focusing method or the preparation of micro-fluidic method; Said albumin is meant one or more in human serum albumin or the bovine serum albumin; Said phospholipid, phospholipid derivant are meant one or more in sphingomyelins, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine, phosphatidyl glycerol, phosphatidylinositols, phosphatidic acid and the derivant thereof, and said high molecular polymer is meant one or more in polylactic acid or the poly lactic coglycolic acid; Said microvesicle particle size range is 0.1-20 μ m, is preferably 0.5-10 μ m.
10. delivering method in the nano-medicament carrier brain according to claim 1 is characterized in that the particle size range of said nano-medicament carrier is 1-1000 nm, is preferably 10-200 nm.
11. Nano medication compositions; Contain nano-medicament carrier and medicine; It is characterized in that said Nano medication compositions also contains microvesicle, said microvesicle is meant that with albumin, phospholipid, phospholipid derivant or high molecular polymer be material; With perfluoropropane, perfluorinated butane or sulfur hexafluoride serves as to fill gas, gets the micron order bubble with cross-linking method, probe ultrasonic method, freeze-drying, emulsion process, mechanical oscillation method, coaxial unidirectional current atomization, flow focusing method or the preparation of micro-fluidic method; Said nano-medicament carrier is meant according to what conventional method prepared can wrap the nanoscale complex that carries and carry medicine; Said medicine is meant maincenter disease treatment and diagnostic medicine; Or pass through the medicine that central action is brought into play the systemic treatment effect; Said drug encapsulation is in nano-medicament carrier; Be in micromolecule chemicals, diagnostic medicine, polypeptide protein medicine and the genomic medicine one or several, said Nano medication compositions is according to claim 1---delivering method targeting drug delivery in 10 arbitrary said brains.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103243073A (en) * 2013-05-13 2013-08-14 东南大学 Carrier system and method for inputting nanoparticles into cell
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
CN105199095A (en) * 2015-09-29 2015-12-30 南京邮电大学 Amphipathic molecular probe based on sulfhydryl substance detection and synthesis method thereof
US20170080114A1 (en) * 2014-03-19 2017-03-23 Imgt Co, Ltd. Dual-Purpose Pat/Ultrasound Contrast Agent Bound with Nanoparticles Containing Drug and Method for Preparing Same
CN107072938A (en) * 2014-11-21 2017-08-18 通用电气公司 For diagnosing the microbubble tethers with treatment use
WO2017157182A1 (en) * 2016-03-14 2017-09-21 杭州普施康生物科技有限公司 Pharmaceutical composition comprising bile salt, preparation method thereof, and application of same
CN108430579A (en) * 2015-11-11 2018-08-21 浩宇生医股份有限公司 The method and kit of brain tumor are treated by using ultrasonic system

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101053810A (en) * 2007-03-01 2007-10-17 上海交通大学 Method for preparing high molecular micro nanometer capsule
CN102137658A (en) * 2008-06-30 2011-07-27 斯兰斯德有限公司 Methods, compositions and systems for local delivery of drugs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101053810A (en) * 2007-03-01 2007-10-17 上海交通大学 Method for preparing high molecular micro nanometer capsule
CN102137658A (en) * 2008-06-30 2011-07-27 斯兰斯德有限公司 Methods, compositions and systems for local delivery of drugs

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* Cited by examiner, † Cited by third party
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US20150343079A1 (en) * 2012-10-25 2015-12-03 Sogang University Research Foundation Ultrasound contrast agent with nanoparticles including drug and method for preparing the same
CN104755108B (en) * 2012-10-25 2018-04-27 艾姆戈特株式会社 With reference to the ultrasonic contrast agents and its manufacture method of the nano-particle containing medicine
CN103243073A (en) * 2013-05-13 2013-08-14 东南大学 Carrier system and method for inputting nanoparticles into cell
US20170080114A1 (en) * 2014-03-19 2017-03-23 Imgt Co, Ltd. Dual-Purpose Pat/Ultrasound Contrast Agent Bound with Nanoparticles Containing Drug and Method for Preparing Same
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
CN107072938A (en) * 2014-11-21 2017-08-18 通用电气公司 For diagnosing the microbubble tethers with treatment use
CN105199095A (en) * 2015-09-29 2015-12-30 南京邮电大学 Amphipathic molecular probe based on sulfhydryl substance detection and synthesis method thereof
CN105199095B (en) * 2015-09-29 2017-05-03 南京邮电大学 Amphipathic molecular probe based on sulfhydryl substance detection and synthesis method thereof
CN108430579A (en) * 2015-11-11 2018-08-21 浩宇生医股份有限公司 The method and kit of brain tumor are treated by using ultrasonic system
WO2017157182A1 (en) * 2016-03-14 2017-09-21 杭州普施康生物科技有限公司 Pharmaceutical composition comprising bile salt, preparation method thereof, and application of same

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