CN106267246A - A kind of ultrasonic in combination targeted microbubble contrast medium and preparation method thereof - Google Patents
A kind of ultrasonic in combination targeted microbubble contrast medium and preparation method thereof Download PDFInfo
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- CN106267246A CN106267246A CN201610652303.0A CN201610652303A CN106267246A CN 106267246 A CN106267246 A CN 106267246A CN 201610652303 A CN201610652303 A CN 201610652303A CN 106267246 A CN106267246 A CN 106267246A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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- General Health & Medical Sciences (AREA)
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- Acoustics & Sound (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Radiology & Medical Imaging (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of ultrasonic in combination targeted microbubble contrast medium and preparation method thereof, ultrasonic in combination targeted microbubble contrast medium, be made up of the component of following concentration: microbubble contrast agent 6～8 × 109Individual/ml, VEGF 5～15mg/ml, anti-ICAM monoclonal antibody 5～15mg/ml and cell-penetrating peptide 0.5～2mg/ml.Preparation method is: VEGF gene and anti-ICAM monoclonal antibody are mixed 1.5～2.5h with microbubble contrast agent by proportioning at 2～4 DEG C by (1)；(2) adding cell-penetrating peptide in step (1) gained mixture, after incubated at room 25～35min, unconjugated cell-penetrating peptide is removed in washing, obtains described ultrasonic in combination targeted microbubble contrast medium.The microbubble contrast agent of the present invention is ultrasonic contrast contrast medium, is again a kind of the most purposive novel gene gene transfection for treating instrument.
Description
Technical field
The present invention relates to a kind of cardiovascular and cerebrovascular diseases medicine, be specifically related to a kind of ultrasonic in combination targeted microbubble contrast medium
And preparation method thereof.
Background technology
Ultrasound medicine technology not only quickly grows at traditional diagnostic field, and also quickly grows in treatment field, special
It it not the hot topic becoming research at present at the gene gene transfection for treating treating cardiac and cerebral vascular diseases of ultrasonic mediation especially.
Changing with dietary habit along with living standards of the people improve, cardiovascular and cerebrovascular disease death has accounted for total dead composition
41%, atherosclerosis causes cardiovascular and cerebrovascular disease to be the major disease that harm human life is healthy.It is implemented with targetedly
Intervene, study effective treatment means, will be to ensureing that people's health is of great importance.
Arterial injury particularly endotheliocyte 26S Proteasome Structure and Function damage is atherosclerosis occur, development initiating because of
Element, and run through atherosclerotic overall process.It is that medicine, intra-arterial prop up currently for atherosclerosis essential therapeutic arsenals
Frame implantation and artery bypass surgical operation therapy.Current medicine such as angiotensin converting enzyme inhibitor, statins,
Calcium ion antagonists etc. can improve inner skin cell function, but these medicines can not make the district of existing endotheliocyte defect
Territory covers endotheliocyte again, and this kind of medicine damages for liver, renal function, and life-time service can cause liver
Disease in terms of dirty, kidney.Current Therapeutic Method still can not reach to recover completely the effect of blood vessel structure and function.How to make
It is a challenging problem that endotheliocyte defect area covers endotheliocyte again, explores in new reparation damaged blood vessels
Chrotoplast, the recovery normal physiological function of endotheliocyte are that the endothelial cell damage angiopathys such as treatment atherosclerosis are anxious
Problem to be solved.Vascular endothelial cell regulation vascular function and vessel homeostasis on play a crucial role, atherosclerosis its
Pathophysiological basis is to cause inner skin cell function to lack of proper care after blood vessel internal membrane damage.After endothelial cell damage, arterial wall middle level is put down
The main cause of Neointimal formation when sliding muscle cell multiplication and extracellular matrix synthesis increase are arterial injury.Repair damage
Endotheliocyte can effectively suppress the formation of new intima, recovers the auterial diastole function that endotheliocyte relies on.Studies have found that
Arterial injury localized transfection vascular endothelial cell growth factor (vascular endothelial growth factor,
VEGF) gene or intravenous injection vegf protein, can make the minimizing of damaged arteries neointimal thickness, arterial endothelial cell rely on
Arterial dilation improve.VEGF can generate, increase vascular permeability and maintain vascular function by induction of vascular, is to have now been found that
The most potent angiogenic factors, VEGFl65It it is the most important phenotype of Angiogensis in 5 kinds of hypotypes.Because
VEGFl65The biological half-life of albumen is short, and costly, the factors such as topical is difficult are difficult to popularization and application.Asahara etc. use
The method of gene therapy, by VEGF165Plasmid is by the damage location of hydrogel sphere ductus bursae Successful transfection to rabbit femoral artery, just
Step achieves the generation of restenosis after minimizing damages.But this transfection is the pressure relied between sacculus and blood vessel wall to be completed,
This pressure may make tunica intima the most impaired, and adds thrombotic risk, leaves latent while treatment
Hidden danger.The most effectively exogenous VEGF is imported internal, make high concentration VEGF orientation be adhered to damaged blood vessels endothelium also
Promote that the new way that vascular endothelial cell proliferation, exploration realize VEGF gene transfection blood vessel wall has important meaning.
Ultrasonic in combination microvesicle promotees gene and transfects:
The method of gene target treatment at present has: viral vector gene treatment, electroporation, medicine couple and perform the operation locally
Inject angiogenesis factor etc., but these methods have applied significant limitation clinically.Contrast-enhanced ultrasound technique in recent years
Make a breakthrough, and microvesicle shell carries specific antibody, gene, medicine, reach targeting diagnosis or therapeutic purposes the most especially
Study hotspot.Ultrasonic provide a kind of non-chemically property, non-viral, Noninvasive for cell dispenser and therapeutic gene
Important means, provides a comparatively ideal Novel noninvasive gene delivery vehicle for people.So-called targeted ultrasound refers to microvesicle
Carrying gene as carrier, when microvesicle arrives particular organization, ultrasonic orientation destroys microvesicle, discharges gene thus reaches at target device
The purpose of official's targeted therapy.Its targeting can by following some realize: 1. microvesicle arrives after target tissue by ultrasonic photograph
The microvesicle penetrated breaks up effect harmony cause porous effect makes gene or medicine be absorbed by target tissue picked-up；2. by tissue specific antibodies
Or part is combined with microbubble surface, thus to target tissue specific adhesion；3. gene or pharmaceutical pack are entrained in microvesicle, be adhered to
Target tissue makes it discharge transfection by ultrasonic explosion microvesicle again.
Ultrasonic in combination microvesicle promotees positive pressure, the DNA of ultrasonic mediation and cell membrane ultrasonic in the mechanism of gene transfection and makees
Play an important role during gene transfects by time lengthening, the cavitation effect of microvesicle and heat effect.Ultrasonic mediation
Microvesicle transfection need meet two conditions: first, active substance can discharge at target region, makes local concentration increase.Second,
The cell membrane of surrounding tissue and microvascular permeability increase.The most this DNA cross-film transfection efficiency can be the lowest.
It is now recognized that ultrasonic in combination microvesicle promotes that gene transfects mainly by cavitation effect produced during microbubble ruptures.
Cavitation effect refers to that the small cavity (cavitation nucleus) being present in liquid is excited under the effect of high strength supersonic, cavitation
Core drastically expands and shrinks until bursting, and during this, cavitation nucleus absorbs substantial amounts of acoustic energy, and by centralized release energy minimum
Region, in core, local temperature and pressure drastically raise, produce therewith powerful shock wave, interior shear force, high speed microbeam jet and from
By quadratic effects such as bases, not only making the microvascular of diameter≤7um, endotheliocyte gap broadening, microvascular permeability increases,
The integrity that can also make cell membrane is destroyed, and produces aperture temporary, reversible, adds membrane passage,
All can strengthen medicine-carrying microvesicle to gene or the transfer of medicine.Meanwhile, ultrasound wave is utilized to smash target in special time and space
Microvesicle in tissue, can improve the targeting for the treatment of.Research finds under the ultrasonic irradiation effect for the treatment of intensity, Ultrasound mediated gene
Transhipment can arrive purpose region accurately, and will not cause acute death or the damage of cell.Research table both domestic and external
Bright, ultrasonic in combination microvesicle can make the transfection of gene and expression substantially increase, it has been reported that the transfection efficiency of naked DNA can be made to improve
3000 times.A1lanLawrie etc. study discovery, the most ultrasonic can strengthen target base after vascular endothelial cell transfection
The expression of cause, additionally, it is observed that therapeutic ultrasound irradiates can suppress the regeneration of vascular smooth muscle, but the activity of Human Umbilical Vein Endothelial Cells
Have no significant effect.Under ultrasonic irradiation, load gene microvesicle targeting ruptures the gene of release and arrives through intercellular substance the most passively
Reaching intracellular, its transfection efficiency is the most extremely limited.
Summary of the invention
The present invention provides a kind of ultrasonic in combination targeted microbubble contrast medium and preparation method thereof, and the microbubble contrast agent of the present invention was both
It is ultrasonic contrast contrast medium, is again a kind of the most purposive novel gene gene transfection for treating instrument.
A kind of ultrasonic in combination targeted microbubble contrast medium, is made up of the component of following concentration:
Said components is dispersed in buffer, is preferably dispersed in the PBS of pH value 7.2-7.4.
Further preferably, it is made up of the component of following concentration:
Most preferably, it is made up of the component of following concentration:
As preferably, described microbubble contrast agent is perfluoropropane human albumin's microvesicle (perfluor shows), human albumin
Octafluoro propane microvesicle (Optison) or sulfur hexafluoride microvesicle (sound Novi).All by city available from (perfluor shows: the quasi-word of traditional Chinese medicines
S20083098, Hu'nan Kangrun Pharmaceutical Co., Ltd.；Optison:FS069, Mallinckralt Medical, St Louis,
MO；SonoVue:BR-1, BRACCO, Milan, Italy).
It is further preferred that described microbubble contrast agent is perfluoropropane human albumin's microvesicle (perfluor shows).Perfluor is aobvious big
Molecule microbubble contrast agent has more preferable pardon compared with for the little molecule microbubble contrast agents such as sound Novi, can be simultaneously by multiple biology
Preparation is dissolved in wherein.
As preferably, described VEGF (VEGF) is VEGF165cDNA, VEGF121cDNA,
VEGF145cDNA or VEGF148cDNA.
VEGF-A is the dimer glycoprotein being combined into disulfide bond crosslinking by two same subunit (Mr 23000), molecule
Amount 40-45KD, its gene is positioned at chromosome 6p21.3, total length 14kb, is made up of 8 exons and 7 introns.Due to it
The montage mode of mRNA is different, forms 7 kinds of existence forms, i.e. VEGF121, VEGF145, VEGF148, VEGF183, VEGF165,
VEGF189, VEGF206, contain 121～206 aminoacid respectively, connect into homodimer with disulfide bond.
VEGF189, VEGF206 contain exon 6, the aminoacid of 7 codings, are combined with heparin, heparin sulfate, extracellular matrix
Energy force rate VEGF145, VEGF165 higher, therefore deadened on the heparin, heparin sulfate of cell surface and extracellular matrix
In, activity in vivo is strong not as VEGF121, VEGF165.
VEGF121, VEGF145, VEGF165 all can inducing cell proliferation and the formation of internal new vessels, great majority
The cell of VEGF expression-A can produce several VEGF-A variant simultaneously, most commonly seen with VEGF121, VEGF165.
Heparin or heparin sulfate can strengthen the affinity of VEGF-A and its receptor.The heparin of VEGF-A and cell surface or born of the same parents
The heparin sulfate proteoglycans of epimatrix combines, and the new vessels factor such as alkaline fiber being releasably stored on Dan Baiduotang proteoglycan PG is female
Cell growth factor (bFGF)；The outer heparin sulfate of born of the same parents can also adjust somatomedin biological activity and with the phase interaction of receptor
With.Then losing the ability combining VEGFR-2 after VEGF121, VEGF165 are oxidized, the heparin sulfate of cell surface can store quilt
The VEGF165 of oxidation, but can not store oxidized VEGF121, and the function of prompting VEGF121 may be strong not as VEGF165, with
Time due in cDNA amplification abundance VEGF165 the highest, so being to apply most a kind of VEGF mono-in clinical and zoopery
Body.
Therefore, consider, described VEGF (VEGF) preferably VEGF165cDNA,
VEGF121cDNA, VEGF145cDNA or VEGF148cDNA；Further preferably, described VEGF (VEGF) base
Because VEGF165cDNA.
VEGF165cDNA contains 441 bases, its base sequence as shown in SEQ ID NO:1, corresponding aminoacid sequence
As shown in SEQ ID NO:2.
As preferably, described anti-ICAM monoclonal antibody is anti-ICAM-1 monoclonal antibody, anti-ICAM-2 monoclonal antibody
Or anti-ICAM-3 monoclonal antibody.Anti-ICAM monoclonal antibody all by city available from, be purchased from the great biotechnology of Shanghai sunrise limited
Company.
As preferably, described cell-penetrating peptide is HIV-1Tat (GRKKRRQRRRPQ (SEQ ID NO:3)), Tat48-60
(GRKKRRQRRRPPQQ (SEQ ID NO:4)), Plsl (RVIRVWFQNKRCKDKK (SEQ ID NO:5)), Transportan
(GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:6)).All obtained by commercial approach, be purchased from the great life of Shanghai sunrise
Thing Science and Technology Ltd..
Cell-penetrating peptide (cell-penetrating peptide, CPP) has the surprising energy carrying macromolecular substances
Power, tool has great advantage: high with the affinity of cell membrane, wears film speed soon, can be degraded rapidly, it is most important that to cell membrane
There is no destructiveness.These are found to be a kind of efficient, safe carrier of development, and actively mediated gene leap membranes barriers is carried out
Gene therapy provides wide prospect, but lacks targeting.By targeted ultrasound microvesicle and cell-penetrating peptide use in conjunction, both will
Learning from other's strong points to offset one's weaknesses, both made cell-penetrating peptide have targeting, the existence of cell-penetrating peptide simultaneously both can be protected, concentrate gene, again can mediated gene
Actively pass biological barrier, improve the transfection efficiency of gene.
Anti-ICAM monoclonal antibody has good targets identification effect to damaged blood vessels inner membrance, in progression of atherosclerosis
During, endothelial injury occurs early than coronary angiography exception and clinical symptoms, and therefore the detection of Endothelial Dysfunction is weight
The early diagnosis index wanted.It is factor-related, predominantly that research shows that vessel endothelium and induced endothelial too much expresses leukocyte with endotheliocyte
Intercellular surface adhesion molecule-1 (ICAM-1) is expressed and is increased.ICAM-1 is a member of cell adhesion molecule immunoglobulin class,
Mainly it is expressed on activated endothelial cells and other antigen mediator cells, mediation endotheliocyte and the Adhesion of leukocyte, promotees
Make endothelial injury.Owing to ICAM-1 expresses the most hardly, so the carrier of anti-ICAM-1 labelling can specific bond
In injured endothelium surface.Microbubble contrast agent is connected with specific antibody, just the available target that can combine with specific receptor
To microvesicle.
The present invention is most preferably combined as: described microbubble contrast agent is that perfluoropropane human albumin's microvesicle is (complete
Fluorine shows)；VEGF (VEGF) gene is VEGF165cDNA；Anti-ICAM monoclonal antibody is anti-ICAM-1 Dan Ke
Grand antibody；Cell-penetrating peptide is HIV-1Tat.
1. microbubble contrast agent preferably employ perfluor show, perfluor shows macromole microbubble contrast agent compared with little molecule microvesicles such as sound Novi
There is for contrast agent more preferable pardon, can multiple biological preparation is dissolved in wherein simultaneously, therefore VEGF165cDNA, anti-
ICAM-1 monoclonal antibody, cell-penetrating peptide-tat can preferably aobvious with perfluor combine；2. VEGF can generate, increase blood by induction of vascular
Pipe permeability and maintenance vascular function, be the most potent angiogenic factors having now been found that, VEGFl65 is in 7 kinds of hypotypes
The most important phenotype of Angiogensis, therefore VEGF165cDNA can more effectively repair damaged blood vessels；The most anti-ICAM-1 is mono-
Clonal antibody can specific bond in damage blood vessel endothelium surface, make ultrasonic in combination microvesicle play the effect of targeted therapy；4. wear
The existence of film peptide-tat both can be protected, concentrate gene, can actively pass biological barrier by mediated gene again, and the transfection improving gene is controlled
Curative effect rate.Thus this kind of ultrasonic in combination targeted microbubble contrast medium can carry out high efficiency gene gene transfection for treating to damaged blood vessels inner membrance,
The therapeutic purposes of cardiovascular and cerebrovascular disease are reached with this.Use the ultrasonic in combination targeted microbubble contrast medium transfection that these components are made
Rate is high, can reach damaged blood vessels inner membrance is carried out high efficiency gene gene transfection for treating, reach effectively controlling of cardiovascular and cerebrovascular disease with this
Treat purpose.
VEGF gene can generate, increase vascular permeability and maintain vascular function by induction of vascular clinically, is to send out at present
The most potent existing angiogenic factors.The microbubble contrast agent of what the present invention obtained be loaded with VEGF gene can pass through ultrasonic one-tenth
As the blood vessel that inner membrance is impaired is detected in real time and diagnoses, it is often more important that the ultrasonic microvesicle release gene that smashes carries out damaged zone
The gene transfection for treating in territory.
The present invention adds what anti-ICAM monoclonal antibody and cell-penetrating peptide were formed to being loaded with in the microbubble contrast agent of VEGF gene
Complex so that the transfection of VEGF gene has more purposiveness, initiative, thus carrys out the high efficiency of real property gene gene transfection for treating.This
Invention provides a kind of new genomic medicine and therapy for the diagnosis and treatment of damaged blood vessels, has bigger clinical meaning.
Present invention also offers the preparation method of a kind of described ultrasonic in combination targeted microbubble contrast medium, comprise the steps of
(1) by VEGF gene and anti-ICAM monoclonal antibody and microbubble contrast agent by proportioning mix at 2～4 DEG C 1.5～
2.5h, obtains mixed liquor, in mixed liquor VEGF mrna concentration be 5～15mg/ml, anti-ICAM MAb concentration be 5～15mg/
Ml, microbubble contrast agent are 6～8 × 109Individual/ml；VEGF gene and anti-ICAM monoclonal antibody attach to microbubble contrast agent shell
Surface forms mixture；
(2) take step (1) gained mixed liquor, add cell-penetrating peptide, incubated at room 25～35min, washing remove not with mix
Thing combine cell-penetrating peptide, be resuspended in buffer, resuspended after mixed liquor identical with taken step (1) mixeding liquid volume, repetition
Operation makes cell-penetrating peptide final concentration reach 0.5～2mg/ml and i.e. obtains ultrasonic in combination targeted microbubble contrast medium.
Washing and resuspended buffer used are PBS, and the pH of PBS is 7.2-7.4.
Add cell-penetrating peptide and hatch rear PBS washing, remove the cell-penetrating peptide not being combined with mixture, i.e. mixture in washing process
Middle VEGF gene and anti-ICAM monoclonal antibody will not run off with microbubble contrast agent, are suspended in PBS after washing, suspend
After volume identical with the volume of the mixed liquor of step (2) taken step (1), detect cell-penetrating peptide concentration, as concentration is below standard, continue
Continuous adding cell-penetrating peptide, wash, suspend, repeatable operation is until cell-penetrating peptide final concentration reaches 0.5～2mg/ml, due in washing process
Do not have the loss of VEGF gene and anti-ICAM monoclonal antibody and microbubble contrast agent, washing terminate resuspended gained mixeding liquid volume with
Taken step (1) mixeding liquid volume is identical, therefore, and VEGF in gained ultrasonic in combination targeted microbubble contrast medium in step (2)
Mrna concentration is 5～15mg/ml, the concentration of anti-ICAM monoclonal antibody is 5～15mg/ml, microbubble contrast agent 6～8 × 109
Individual/ml, cell-penetrating peptide concentration are 0.5～2mg/ml.
It is further preferred that in the mixed liquor obtained of step (1) VEGF mrna concentration be 10～15mg/ml, anti-ICAM
The concentration of monoclonal antibody is 5～10mg/ml, microbubble contrast agent concentration is 6～8 × 109Individual/ml, adds in step (2) and wears film
After peptide, cell-penetrating peptide final concentration reaches 0.5～1.5mg/ml；It is further preferred that VEGF in the mixture obtained of step (1)
Mrna concentration is 12mg/ml, the concentration of anti-ICAM monoclonal antibody is 9mg/ml, microbubble contrast agent is 7 × 109Individual/ml, step
(2) after adding cell-penetrating peptide in, cell-penetrating peptide final concentration reaches 1mg/ml.
The preparation method of the present invention, most preferably, comprises the steps:
(1) VEGF gene, anti-ICAM monoclonal antibody are mixed with the volume ratio of 1:1:3 with microbubble contrast agent at 4 DEG C
Closing 2h and obtain mixed liquor, in mixed liquor, VEGF mrna concentration is 12mg/ml, and the concentration of anti-ICAM monoclonal antibody is 9mg/ml, micro-
Bubble contrast agent is 7 × 109Individual/ml；The shell that VEGF gene and anti-ICAM monoclonal antibody attach to microbubble contrast agent is formed mixed
Compound；
(2) take step (1) gained mixed liquor, add cell-penetrating peptide, incubated at room 30min, PBS washing 5min totally three times, wash
Wash and remove the cell-penetrating peptide that is not combined with mixture, be resuspended in buffer after washing every time, resuspended after mixed liquor and taken step
Suddenly (1) mixeding liquid volume is identical, and cell-penetrating peptide final concentration reaches 1mg/ml and i.e. obtains ultrasonic in combination targeted microbubble contrast medium.
The present invention is prepared for the microbubble contrast agent of a kind of VEGF gene target treatment damaged blood vessels inner membrance, and have employed tool
The cell-penetrating peptide actively transfecting function is had to transfect promoting VEGF gene to carry out actively.
Detailed description of the invention
Example below is used for further illustrating the present invention, but limits the content that the present invention relates to the most in any form.
Embodiment 1
Perfluor shows 7 × 109Individual/ml
VEGF165cDNA 12mg/ml
Anti-ICAM-1 monoclonal antibody 9mg/ml
Cell-penetrating peptide (HIV-1Tat) 1mg/ml
VEGF165cDNA gene and anti-ICAM-1 monoclonal antibody are shown the volume ratio with 1:1:3 at 4 DEG C with perfluor
Mixing 2h obtain mixed liquor, in mixed liquor VEGF165cDNA mrna concentration be 12mg/ml, the concentration of anti-ICAM-1 monoclonal antibody
7 × 10 are shown for 9mg/ml, perfluor9Individual/ml, VEGF165cDNA gene and anti-ICAM-1 monoclonal antibody attach to microvesicle radiography
The mixture that the shell of agent is formed.
Obtained by mixed liquor in add after cell-penetrating peptide (HIV-1Tat) incubated at room 30min, PBS washing 5min totally three
Secondary, remove unconjugated cell-penetrating peptide, be resuspended in PBS after washing every time, resuspended after mixed liquor mixed with taken
Close liquid and amass identical, make cell-penetrating peptide final concentration reach 1mg/ml, i.e. can be made into product.
Embodiment 2
Perfluor shows 7 × 109Individual/ml
VEGF165cDNA 12mg/ml
Anti-ICAM-1 monoclonal antibody 9mg/ml
VEGF165cDNA gene and anti-ICAM-1 monoclonal antibody are shown the volume ratio with 1:1:3 at 4 DEG C with perfluor
Mixing 2h obtain mixed liquor, in mixed liquor VEGF165cDNA mrna concentration be 12mg/ml, the concentration of anti-ICAM-1 monoclonal antibody
7 × 10 are shown for 9mg/ml, perfluor9Individual/ml, VEGF165cDNA gene and anti-ICAM-1 monoclonal antibody attach to microvesicle radiography
The formed mixture of shell of agent.
Embodiment 3
Perfluor shows 7 × 109Individual/ml
VEGF165cDNA12mg/ml
Cell-penetrating peptide (HIV-1Tat) 1mg/ml
At 4 DEG C, mix 2h with the volume ratio of 1:3 by aobvious to VEGF165cDNA gene and perfluor, obtain mixed liquor, mixed liquor
Middle VEGF165cDNA mrna concentration is the shell formation mixing that 12mg/ml, VEGF165cDNA gene attaches to microbubble contrast agent
Thing.
Obtained by mixed liquor in add after cell-penetrating peptide (HIV-1Tat) incubated at room 30min, PBS washing 5min totally three
Secondary, remove unconjugated cell-penetrating peptide, be resuspended in PBS after washing every time, resuspended after mixed liquor mixed with taken
Close liquid and amass identical, make cell-penetrating peptide final concentration reach 1mg/ml, i.e. can be made into product.
Embodiment 4VEGF165cDNA gene transfection efficiency measures
Embodiment 1, embodiment 2, each 1ml of embodiment 3 are separately added into FITC labelling, insert 37 DEG C of calorstats, PBS after 1h
Wash 4 times, each 5min, inject the embodiment 1 of FITC labelling, embodiment by modeling successful new zealand white rabbit auricular vein
2, embodiment 3, then use in White Rabbit aortic segment ultrasonic break up embodiment 1, embodiment 2, embodiment 3 prepare finished product microvesicle
Discharge gene, put to death new zealand white rabbit after 14d and carry out dissecting and intercept aortic segment blood vessel and make specimen, finally carry out altogether
Confocal laser microscope is observed.After FITC labelling, visible specific green in the vascular endothelial cell of VEGF expression 165cDNA
Color fluorescence, the cell number in 10 250 times of visuals field of counting and the positive expression cell number in each visual field, the ratio of the two is transfection
Rate.
The transfection efficiency recording embodiment 1 is (46.32 ± 2.81) %；Record the transfection efficiency of embodiment 2 for (31.26 ±
3.17) %；The transfection efficiency recording embodiment 3 is (24.13 ± 4.25) %.
The present embodiment animal experimental model is: set up the animal model of ventral aorta inner film injury: healthy New Zealand DABAI
Rabbit, average body quality 2.2kg, give 1% cholesterol particles 2g every day and feed 4 weeks, use high fat diet injured blood vessel endothelium.
The zoopery of embodiment 5 ultrasonic in combination targeted microbubble contrast medium mediation Cerebral Infarction Treatment
60 white rabbits after modeling successfully are randomly divided into test A group, test B group, matched group, and often group 20, adopts respectively
Differently treat.1. test A group uses embodiment 1, is injected by white rabbit auricular vein, and original measurement is that 1ml is left
The right side, is administered 0.5ml in the most every about 4 minutes, and in addition to first administration, doses at intervals 8 times, doses at intervals adds up 4ml；Use simultaneously
Ultrasound mediated therapy, instrument is chosen Philips iU 22 color Doppler sonography diagnostic apparatus, is enabled second harmonic pattern, and incite somebody to action
Tranmitting frequency and accept frequency and be adjusted to 1.5MHz, 3.5MHz respectively, is adjusted to maximum by mechanical index, ultrasound intensity be 2 to
2.5W/cm2.2. test B group employing Ai Tongli intravenous injection, using dosage 0.9mg/kg, front 1/10 dosage intravenous injection immediately,
The slowly at the uniform velocity intravenous injection of doses remaining medicine.3. matched group uses saline i injection, concrete injecting method and consumption
Identical with test A group.Three groups all after treatment 14d white rabbit is carried out coherence check, and put to death white rabbit.Result this test cerebral infarction
District's quality accounting is respectively A group 13.8 ± 3.2, B group 18.8 ± 4.1ab, matched group 25.1 ± 4.6a, hence it is evident that be better than testing B group
And matched group (P ＜ 0.05), illustrate that giving cell-penetrating peptide-tat mediation VEGF165cDNA gene carries out gene transfection well,
May advantageously facilitate the regeneration of infarction vascular endothelial cell, and promote to generate new Doppler flow mapping blood vessel, improve infarcted region blood supply,
Improve cerebral anoxia situation.
The gene transfection efficiency of embodiment 6 variable concentrations component measures
12 white rabbits after modeling successfully are randomly divided into experiment A group, and (perfluor shows: 6 × 109Individual/ml, VEGF165cDNA:
10mg/ml, anti-ICAM-1 monoclonal antibody: 5mg/ml, cell-penetrating peptide (HIV-1Tat): 0.5mg/ml), B group (perfluor show: 7 ×
109Individual/ml, VEGF165cDNA:12mg/ml, anti-ICAM-1 monoclonal antibody: 9mg/ml, cell-penetrating peptide (HIV-1Tat): 1mg/
Ml), (perfluor shows C group: 8 × 109Individual/ml, VEGF165cDNA:15mg/ml, anti-ICAM-1 monoclonal antibody: 10mg/ml, wear
Film peptide (HIV-1Tat): 1.5mg/ml), often group 4, is respectively adopted variable concentrations component and carries out transfection efficiency mensuration.By A group, B
Group, each 1ml of C group are separately added into FITC labelling, insert 37 DEG C of calorstats, and after 1h, PBS washes 4 times, and each 5min is big by New Zealand
White rabbit auricular vein injects the A group of FITC labelling, B group, C group, then uses in White Rabbit aortic segment and ultrasonic break up microvesicle
Release gene, after 14d put to death new zealand white rabbit carry out dissect intercept aortic segment blood vessel make specimen, finally carry out copolymerization
Burnt laser capture microdissection sem observation.After FITC labelling, visible specific green in the vascular endothelial cell of VEGF expression 165cDNA
Fluorescence, the cell number in 10 250 times of visuals field of counting and the positive expression cell number in each visual field, the ratio of the two is transfection efficiency.
The transfection efficiency recording A group respectively is (32.61 ± 3.12) %；The transfection efficiency of B group is (46.32 ± 2.81) %；C group
Transfection efficiency is (33.57 ± 4.28) %；B group transfection optimum for transfection efficiency, therefore B group concentration is configured to optimal concentration configuration.
The present embodiment animal experimental model is: set up the animal model of ventral aorta inner film injury: healthy New Zealand DABAI
Rabbit, average body quality 2.2kg, give 1% cholesterol particles 2g every day and feed 4 weeks, use high fat diet injured blood vessel endothelium.
The foregoing is only the case that is embodied as of patent of the present invention, but the technical characteristic of patent of the present invention is not limited to
This, any those skilled in the relevant art in the field of the invention, the change made or modify all contain in the present invention special
Among profit scope.
Claims (8)
1. a ultrasonic in combination targeted microbubble contrast medium, it is characterised in that be made up of the component of following concentration:
Ultrasonic in combination targeted microbubble contrast medium the most according to claim 1, it is characterised in that described microbubble contrast agent is perfluor
Propane human albumin's microvesicle, human albumin's octafluoro propane microvesicle or sulfur hexafluoride microvesicle.
Ultrasonic in combination targeted microbubble contrast medium the most according to claim 1, it is characterised in that described microbubble contrast agent is perfluor
Propane human albumin's microvesicle.
Ultrasonic in combination targeted microbubble contrast medium the most according to claim 1, it is characterised in that described VEGF gene is
VEGF165cDNA, VEGF121cDNA, VEGF145cDNA or VEGF148cDNA.
Ultrasonic in combination targeted microbubble contrast medium the most according to claim 1, it is characterised in that described VEGF gene is
VEGF165cDNA。
Ultrasonic in combination targeted microbubble contrast medium the most according to claim 1, it is characterised in that described anti-ICAM monoclonal antibody
For anti-ICAM-1 monoclonal antibody, anti-ICAM-2 monoclonal antibody or anti-ICAM-3 monoclonal antibody.
Ultrasonic in combination targeted microbubble contrast medium the most according to claim 1, it is characterised in that described cell-penetrating peptide is HIV-
1Tat, Tat48-60, Plsl or Transportan.
8. the preparation method of a ultrasonic in combination targeted microbubble contrast medium, it is characterised in that comprise the steps:
(1) VEGF gene and anti-ICAM monoclonal antibody are mixed 1.5～2.5h with microbubble contrast agent by proportioning at 2～4 DEG C
Mixed liquor, in mixed liquor VEGF mrna concentration be 5～15mg/ml, anti-ICAM MAb concentration be 5～15mg/ml, micro-
Bubble contrast concentration is 6～8 × 109Individual/ml；VEGF gene and anti-ICAM monoclonal antibody attach to microbubble contrast agent shell table
Face forms mixture；
(2) taking step (1) gained mixed liquor, add cell-penetrating peptide, incubated at room 25～35min, washing is removed and is not tied with mixture
Close cell-penetrating peptide, be resuspended in buffer, resuspended after mixed liquor identical with taken step (1) mixeding liquid volume, repetitive operation
Make cell-penetrating peptide final concentration reach 0.5～2mg/ml and i.e. obtain ultrasonic in combination targeted microbubble contrast medium.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1579554A (en) * | 2004-05-18 | 2005-02-16 | 浙江大学医学院附属第一医院 | Gene-carrying micro-bublle of target transfergene, its preparation method and use |
| CN1814305A (en) * | 2005-12-09 | 2006-08-09 | 重庆医科大学 | Gene-ordrug-carrying-carrying ultrasonic microvesicle contrast-media and preparing method thereof |
| WO2006129080A1 (en) * | 2005-05-31 | 2006-12-07 | The Institute Of Cancer Research: Royal Cancer Hospital | Materials and methods for transducing cells with a viral vector |
| CN101028524A (en) * | 2006-03-03 | 2007-09-05 | 重庆融海超声医学工程研究中心有限公司 | Supersonic microvesicle target positioning controlled-release/gene device and target transferring method |
-
2016
- 2016-08-10 CN CN201610652303.0A patent/CN106267246A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1579554A (en) * | 2004-05-18 | 2005-02-16 | 浙江大学医学院附属第一医院 | Gene-carrying micro-bublle of target transfergene, its preparation method and use |
| WO2006129080A1 (en) * | 2005-05-31 | 2006-12-07 | The Institute Of Cancer Research: Royal Cancer Hospital | Materials and methods for transducing cells with a viral vector |
| CN1814305A (en) * | 2005-12-09 | 2006-08-09 | 重庆医科大学 | Gene-ordrug-carrying-carrying ultrasonic microvesicle contrast-media and preparing method thereof |
| CN101028524A (en) * | 2006-03-03 | 2007-09-05 | 重庆融海超声医学工程研究中心有限公司 | Supersonic microvesicle target positioning controlled-release/gene device and target transferring method |
Non-Patent Citations (6)
| Title |
|---|
| 任建丽 等.: "超声微泡联合穿膜肽促基因转染治疗急性心肌梗死的动物实验研究", 《中国超声医学杂志》 * |
| 凌智瑜 等.: "超声微泡造影剂介导V EGF 基因治疗大鼠心肌缺血的实验性研究", 《中国超声医学杂志》 * |
| 刘克辛 编著.: "《肝细胞增殖因子(HGF)分子医学研究最新进展》", 31 January 2001, 天津科学技术出版社 * |
| 孙泉 等.: "超声介导靶向微泡治疗脑梗死白兔动物实验研究", 《中华全科医学》 * |
| 蔡丽萍 等.: "靶向超声介导VEGF165cDNA基因治疗大鼠心肌缺血的实验研究", 《中华超声影像学杂志》 * |
| 蔡丽萍.: "靶向超声介导血管内皮165生长基因及抗细胞间粘附分子-1单克隆抗体协同治疗大鼠心肌缺血的实验性研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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