CN108114291A - Target multi-modal imaging nano-contrast agent of inversion of phases and preparation method thereof - Google Patents
Target multi-modal imaging nano-contrast agent of inversion of phases and preparation method thereof Download PDFInfo
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- CN108114291A CN108114291A CN201711228734.5A CN201711228734A CN108114291A CN 108114291 A CN108114291 A CN 108114291A CN 201711228734 A CN201711228734 A CN 201711228734A CN 108114291 A CN108114291 A CN 108114291A
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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
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
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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
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
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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
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
- A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
-
- 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
- A61K49/225—Microparticles, microcapsules
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
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- Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Radiology & Medical Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Nanotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- Acoustics & Sound (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses the targeting multi-modal imaging nano-contrast agents of inversion of phases, including lipid shell membrane, oleic acid oxidation iron nano-particle and drug HCPT are mounted on lipid shell membrane, and is modified with folic acid on lipid shell membrane.The technical problem to be solved in the present invention is to provide one kind under the action of low-strength focusing ultrasonic, liquid-gas phase transition, enhancing development occurs, and discharges drug, realizes multi-modal imaging and precisely treatment to tumour.
Description
Technical field
The present invention relates to ultrasonic image fields, and in particular to it is a kind of targeting inversion of phases it is multi-modal imaging nano-contrast agent and its
Preparation method.
Background technology
Traditional single image check mode is respectively provided with respective deficiency, is had some limitations using upper, and more
Kind visual fusion imaging so that various image check means are learnt from other's strong points to offset one's weaknesses, and are had complementary advantages, and can improve the accuracy of diagnosis.Fusion
The multi-functional contrast agent of two or more imaging patterns is the research hotspot of modern molecular iconography.Multi-modal contrast agent except
The function of imaging, additionally it is possible to load drug, gene etc., under the guiding and monitoring of image, realize controllable targeted therapy.Cause
This, multi-modal multi-functional contrast agent can integrate imaging with treatment, for disease getting up early Accurate Diagnosis and precisely controllably control
Treatment is taken a firm foundation.
Ultrasound microbubble contrast agent provides help for the diagnosis of disease, but common ultrasonic microbubble cannot meet molecular image
Learn the demand to molecular probe.General a few micrometers of the sizes of microbubble contrast agent, and tumour capillary endothelium gap about 100-
780nm, microvesicle are difficult to reach tissue space through blood vessel endothelium, limit application of the microvesicle as ultrasound molecular probe.Have
Nano grade air bubbles i.e. Na Pao is made in gas microbubbles volume-diminished by person, but because its backscattering ability is weaker, enhances ultrasonic development
Ability it is limited.Due to the impact of intravascular blood flow so that the ability that targeted microbubble is combined with vascular targeting reduces.Microvesicle
Half-life short (3-15 minutes) in vivo, also limits it and further applies.In order to overcome microvesicle as ultrasound molecular probe
Deficiency, inventor design it is a variety of package liquid fluorocarbons nanoparticles.Liquid fluorocarbon possesses some properties, such as highly dense
Degree, high gas solubility, high diffusivity coefficient, low surface tension are low, low viscosity, hypotoxicity, hydrophobicity, oleophobic, preferable oxygen carrying
Ability, thermal inertia, x-ray impermeability etc. are laid a good foundation for its application in the field of medicine.Lanza team uses phosphorus
Fat material and liquid fluorocarbon PFOB prepare liquid fluorine carbon-nano lipid microsphere, uniform in size, grain size using high-pressure homogeneous method
Less than 200nm, facilitate penetration of tumour capillary endothelium gap and reach outside blood vessel, when the nano-lipid microsphere aggregation to target organ
The development of gray scale ultrasound can be enhanced afterwards.However liquid fluorocarbon PFOB carrys out the effect of Enhance ultrasonography not very by building-up effect
Ideal can not show a candle to the harmonic imaging of ultrasonic microbubble.Liquid fluorocarbon nanoparticle volume is small, can pass through capillary endothelium gap
And reaching tissue, microballoon is stablized compared with microvesicle property, can cycle the long period in vivo.But because microballoon core is liquid,
It is very weak to the reflex of ultrasound, it is impossible to effectively to enhance ultrasonic development.Various Medical Imaging Technologies such as ultrasound, optoacoustic, magnetic
Resonance, CT etc. respectively have excellent lack in tissue penetration, image resolution ratio, inspection susceptibility, image taking speed and inspection fee etc.
Point.Current various Imaging Techniques have respective image enhancing agents.Although reinforcing agent provides help to the diagnosis and treatment of disease,
But still the limitation of single image check cannot be broken through.With making constant progress for Medical Imaging, seek multi-modal more work(
Energy contrast agent improves the accuracy of image diagnosis and treatment, becomes grinding for modern medicine so as to fulfill the mutual supplement with each other's advantages of a variety of image technologies
Study carefully emphasis.
The content of the invention
The technical problem to be solved in the present invention is to provide one kind under the action of low-strength focusing ultrasonic, liquid gas phase occurs
Become, enhancing development, and discharge drug, realize multi-modal imaging and precisely treatment to tumour.
In order to solve the above technical problem, the present invention provides following technical solutions：Target the multi-modal imaging nanometer of inversion of phases
Contrast agent including lipid shell membrane, is mounted with oleic acid oxidation iron nano-particle and drug HCPT on lipid shell membrane, and on lipid shell membrane
It is modified with folic acid.
Further, it is enclosed with liquid fluorocarbon inside lipid shell membrane.
Further, grain size is 321 ± 67nm, and grain size polydispersity index PDI is 0.43.
Further, ZETA current potentials are -50mV.
Further, HCPT envelop rates are 60.51% ± 2.33%, and drugloading rate is 8.33% ± 0.57%.
The targeting multi-modal imaging nano-contrast agent of inversion of phases using the present invention, Malvern instruments measure receiving for the present invention
Grain of rice grain size about 321 ± 67nm, nanoparticle of the invention can pass through tumour capillary endothelium gap (100nm-780nm),
So it disclosure satisfy that requirement of the present invention to its grain size；Malvern instrument measures its Zeta potential as -50mV, can ensure nanometer
Grain relative stability and will not rapid precipitation.The morphologic detections knot such as light microscopic, fluorescence microscope and laser confocal microscope
Fruit shows, FA/Fe3O4/ PFP/HCPT Lipid nanoparticle good dispersions, uniform in size, into small spheroidal or dotted, these are special
Property is all beneficial to nanoparticle and passes through tumour capillary endothelium gap and reach near tumor cells.Atomic absorption spectrum
Method measures the FA/Fe of various concentration3O4/ PFP/HCPT@Lipid nanoparticles correspond to the iron ion of various concentration.
FA/Fe of the present invention3O4/ PFP/HCPT Lipid contrast agent can stablize under the conditions of 4 DEG C preserve 48 it is small when.Many institute's weeks
Know, about 29 DEG C of PFP boiling points are in a liquid state under room temperature, and (37 DEG C) gasifications are in gaseous state under physiological temp.But it is rolled into and receives via phosphatide
After scale particles, because the Laplace pressure increase being applied to around nanoparticle, PFP gasifications threshold value will raise.Present invention hair
Existing, at 40 DEG C, nanoparticle is not undergone phase transition substantially, and when temperature of heating plate is gradually risen up to 50 DEG C, nanoparticle phase transformation is just most
Significantly, see that the bubble generated after phase transformation is most under microscope.High performance liquid chromatography measure HCPT envelop rates for 60.51% ±
2.33%, drugloading rate is 8.33% ± 0.57%.Drug release test shows that HCPT is avoided in slowly release in nanoparticle
The issuable adverse reaction of burst release.One of the factor of ultrasound as most effective promotion liquid fluorocarbon phase transformation, in the present invention
It is excited PFP phase transformations with low-strength focusing ultrasonic and is promoted FA/Fe3O4/ PFP/HCPT@Lipid discharge drug.The present invention is made
Phospholipid composition is identical with the cell membrane phospholipid ingredient of biologic artifact, without any side effects to organism.Liquid fluorocarbon
Because having good oxygen carrying capacity, it can be used as blood substitute, possess biological safety, and liquid fluorocarbon energy disperse is dissolved in
Blood is excreted by exhaling, and is not involved in vivo biochemical degradation.Fe3O4Biological safety is high, in vivo by red thin
Normal plasma iron pond can be entered after born of the same parents' metabolism, participate in organism metabolism process.
There is liquid fluorocarbon PFP good thermal induced phase transition and sound to cause phase-change characteristic, and Superparamagnetic Iron Oxide Fe3O4 possesses
Enhance the characteristic of optoacoustic and Magnetic Resonance Imaging, PFP and Fe3O4It is loaded into a nano-carrier, can reach multi-modal aobvious simultaneously
The purpose of picture.In conjunction with drug HCPT, which will realize multi-modal and multi-functional requirement, become great potential
Molecular probe.
The present invention also provides another technical solution, the preparation method of the multi-modal imaging nano-contrast agent of targeting inversion of phases,
It is prepared using rotary evaporation-ultrasonic method.
Further, the operating procedure of rotary evaporation-ultrasonic method is as follows：
1), dissolve：First by oleic acid oxidation iron nano-particle, drug HCPT and the fat for including phosphatide-folic acid synthesis material
After mass shell film raw material is dissolved with organic solvent；
2), rotary evaporation：Decompression rotary evaporation, which is carried out, in Rotary Evaporators removes organic solvent；
3), it is cleaned by ultrasonic：It is placed in again in ultrasonic cleaning agent and cleans vibration；
4), centrifuge：4 DEG C of refrigerators of postposition are centrifuged, obtain sample.
Further, after the ultrasonic cleaning of step 3), further included step 3a), add in liquid fluorocarbon, using sound and vibration instrument carry out
After emulsification, then the operation by emulsifying liquid progress step 4).
Further, the solution temperature in the step 1) is 50 DEG C；
The rotating speed of Rotary Evaporators in the step 2) is rotating speed：80rpm, when the time is 2 small；
The step 3a) in the whole ice bath of sound and vibration instrument emulsification, and power is 100w, time 6min；
The temperature centrifuged in the step 4) is 4 DEG C, rotating speed 6000rpm, time 5min, is centrifuged altogether three times.
Further, step 3a) in sound and vibration instrument by the way of sound and vibration is interrupted.
The present invention is using rotary evaporation-ultrasonic method, and by the superparamagnetic iron oxide particle of oleic acid modified, (main component is
Fe3O4), liquid fluorocarbon PFP and drug HCPT wrap up into the lipidic shell being made of a variety of phospholipid compositions and cholesterol, to grind
FA/Fe processed3O4/ PFP/HCPT@Lipid nanoparticles.First by lipid, Fe3O4Particle and drug HCPT are dissolved in organic solvent
In (mixed solvent of chloroform and methanol), dissolving must assure that the sufficient time or using heating or the side of sound and vibration dissolution
Formula so that solute fully dissolves, and is otherwise difficult to form uniform and stable FA/Fe3O4/ PFP/HCPT@Lipid nanoparticles.It adds in
The process whole process ice bath of PFP drops and sound and vibration, the amount of heat otherwise formed during sound and vibration easily promote PFP phase transformations, take
Be be interrupted sound and vibration mode (on:5s,off:5s), so that the heat that sound and vibration generates can in time dissipate in the off phases.
Description of the drawings
Fig. 1 is the structure diagram of the multi-modal imaging nano-contrast agent of present invention targeting inversion of phases.
Fig. 2 is the multi-modal imaging nano-contrast agent of present invention targeting inversion of phases in the single nanoparticle Electronic Speculum of transmission electron microscope observing
Figure.
Fig. 3 is the external enhancing photoacoustic imaging experiment of the multi-modal imaging nano-contrast agent of present invention targeting inversion of phases
Fe3O4、FA/Fe3O4/ PFP/HCPT@Lipid, FA/PFP/HCPT@Lipid optoacoustic figures.
Fig. 4 is in the external enhancing magnetic resonance imaging experiment of the multi-modal imaging nano-contrast agent of present invention targeting inversion of phases
The external magnetic resonance image of I-Ⅻ group.
Fig. 5 is the multi-modal imaging nano-contrast agent of present invention targeting inversion of phases in the targeted therapy effect experiment of oophoroma
Nude mouse tumor growth curve chart.
Specific embodiment
First, the multi-modal imaging nano-contrast agent (FA/Fe of present invention targeting inversion of phases3O4/ PFP/HCPT@Lipid), tool
The preparation method of body is：
1), dissolve：By HSPC- hydrogenated soy phosphatidyl cholines (Avanti companies of the U.S.), DSPE (PEG2000) Folate- phosphorus
(U.S. Avanti is public for fat PEG folic acid (Chongqing Pu Nuowei bio tech ltd), DPPG-1,2- palmityls phosphatidyl glycerol
Department), CHOL- cholesterol (Sangon Biotech (Shanghai) Co., Ltd.) and HCPT be according to mass ratio 10:4:3:3:2 nominals
Common 22mg is taken to add in round-bottomed flask, while adds in 100 μ L oleic acid oxidation iron nano-particles OA-Fe3O4(25mg/mL) measures 10mL
Chloroform and 10mL methanol add in round-bottomed flask, close round-bottomed flask mouth, insert in 50 DEG C of water and be heated to fully dissolving.
2), rotary evaporation：Round-bottomed flask is fixed on Rotary Evaporators (temperature after dissolving 20min：50 DEG C) subtracted
Rotary evaporation is pressed to remove organic solvent, rotating speed：80rpm, time：2 it is small when, formed one layer of uniform brown it is thin
Film.
3), it is cleaned by ultrasonic：Round-bottomed flask is removed after evaporation, 4mL PBS vibrations, hydration is added in, then burns round bottom
Bottle, which is placed in supersonic wave cleaning machine, cleans vibration, until round-bottomed flask inner wall brown film separation obtains brown suspension, it will be mixed
Suspension is transferred in 10mL EP pipes.
4) PFP emulsifications, are added in：Under whole condition of ice bath, 200 μ LPFP are added dropwise into EP pipes, using sound and vibration instrument pair
Suspension is emulsified in EP pipes, sound and vibration instrument power in emulsion process：100w, time：6min(on:5s,off:5s).
5), centrifuge：Emulsion liquid obtained is placed in high speed freezing centrifuge and is centrifuged, temperature：4 DEG C, turn
Speed：6000rpm, time：5min, three times, it is spare that gained sample puts 4 DEG C of refrigerators for centrifugation altogether.
The obtained multi-modal imaging nano-contrast agent of present invention targeting inversion of phases is as shown in Figure 1, including lipid shell membrane, fat
Liquid fluorocarbon is enclosed with inside mass shell film, oleic acid oxidation iron nano-particle and drug HCPT, and lipid are mounted on lipid shell membrane
Folic acid is modified on shell membrane.
2nd, the characteristic and performance of the multi-modal imaging nano-contrast agent of inversion of phases are targeted
1) characteristic of the multi-modal imaging nano-contrast agent of inversion of phases is targeted
(1) after being dissolved with PBS, FA/Fe3O4/ PFP/HCPT@Lipid appearances are brown, stand without apparent layering.Under light microscopic
Observation, FA/Fe3O4/ PFP/HCPT@Lipid are spherical in shape, are evenly distributed, and size is homogeneous.Fluorescence microscope and laser co-focusing are shown
Micro- Microscopic observation, FA/Fe3O4/ PFP/HCPT@Lipid are spherical in shape or dotted, are evenly distributed, and size is more uniform.
(2) as shown in Fig. 2, the single FA/Fe of transmission electron microscope observing3O4/ PFP/HCPT Lipid nanoparticles are in black ball shape knot
Structure, form rule.
(3) Malvern laser particle instruments detect FA/Fe3O4/ PFP/HCPT@Lipid 321 ± 67nm of grain size, grain size are more
Dispersion index (PDI) is 0.43, and Zeta potential is -50mV.
(4) Atomic Absorption Spectrometry obtains the FA/Fe of various concentration3O4/ PFP/HCPT@Lipid nano-emulsions (5 μ g mL-1,
10μg mL-1, 20 μ g mL-1, 40 μ g mL-1, 80 μ g mL-1, 160 μ g mL-1, 320 μ g mL-1, 640 μ g mL-1, 1280 μ g
mL-1,2560μg mL-1) in Fe concentration be respectively (0.43 ± 0.08 μ g mL-1, 0.71 ± 0.09 μ g mL-1, 1.63 ± 0.31
μg mL-1, 3.52 ± 1.21 μ g mL-1, 7.60 ± 1.35 μ g mL-1, 15.05 ± 3.16 μ g mL-1, 29.12 ± 4.05 μ g mL-1, 53.32 ± 5.25 μ g mL-1, 117.65 ± 6.36 μ g mL-1, 236.37 ± 11.97 μ g mL-1)。
2) Detection of Stability of the multi-modal imaging nano-contrast agent of inversion of phases, is targeted
Testing result is：Under the conditions of 4 DEG C, nanoparticle grain size within 48 hours without significantly increasing, 48 it is small when after grain size delay
Slow increase.
3), the multi-modal imaging nano-contrast agent thermal induced phase transition of light Microscopic observation targeting inversion of phases
Testing result is：Nanoparticle kept stable at 40 DEG C, nanoparticle grain size is more uniform under light microscopic, has no apparent phase
Become, liquid-gas phase transition occurs for part nanoparticle at 45 DEG C, volume increase, and phase transformation nanoparticle quantity showed increased at 50 DEG C, volume increases
Greatly, 55 DEG C when only deposit the bubble after a small amount of phase transformation.
4) measure of HCPT contents in the multi-modal imaging nano-contrast agent of inversion of phases, is targeted
Testing result is：HCPT calibration curve equations are：Y=106.8X+1560R=0.999 shows HCPT in 20-
250ng mL-1It is good with peak area linear relationship in concentration range.HCPT envelop rates are 60.51% ± 2.33%, and drugloading rate is
8.33% ± 0.57%, corresponding FA/Fe3O4Drug concentration in/PFP/HCPT@Lipid solution is about 330.24 ± 65.49 μ
g mL-1。
5) the multi-modal imaging nano-radiography agent emulsion drug release of inversion of phases, is targeted
Testing result is：Drug release test shows, FA/Fe3O4Drug HCPT in/PFP/HCPT@Lipid lotions is in
Slowly release, 24 it is small when release about 25%, 48 it is small when release about 60%, 60 it is small when after drug release flatten out it is slow.
6), the detection of the multi-modal imaging nano-radiography agent emulsion controlled release drug of LIFU irradiation targeting inversion of phases
Testing result is：With the increase of LIFU intensity, release amount of medicine is also with increase.0.8W/cm2、2min,
1.6W/cm2、2min,2.4W/cm2, 2min and 3.2W/cm2, release amount of medicine corresponding to 2min respectively about 3.2% ±,
11%th, 27%, 56%.
3rd, FA/Fe3O4The targeting experiment of/PFP/HCPT@Lipid contrast agent
Using oophoroma SKOV3 cell lines, using situation is targeted in model of nude mice bearing tumor observation nanoparticle body, it is divided into folic acid
Targeting group (present invention), non-targeted group and antagonism group.
Experimental result is：
1st, the external target-seeking experimental study of nanoparticle
After fluorescent dye DAPI dyeing, nucleus is under laser confocal microscope in blueness；It is contaminated through fluorescent dye DiO
After color, cell membrane is under laser confocal microscope in green；After fluorescent dye DiI dyeing, nanoparticle is in laser co-focusing
It takes on a red color under microscope.In folacin receptor targeting group, it is seen that the red fluorescence of more representative targeted nano granule is gathered in cell
Around film, and apparent red fluorescence aggregation is had no around non-targeted group and antagonism group, cell membrane.
2nd, target-seeking experimental study in nanoparticle body
The result shows that：For folate-targeted group nude mice after targeted nano lotion 0.5h is injected, tumor by local is visible to represent targeting
The red fluorescence of nanoparticle, until 1h, the increase of red fluorescence scope, are gradually reduced to 6h and 12h extent of fluorescence.And non-targeted group is swollen
Knurl is local in being showed no apparent red fluorescence aggregation in entire observing time.It is shown in targeting group and non-targeted group of nude mice liver
Stronger red fluorescence.In vitro fluorescence experiments are shown after injection nanoparticle 12h, targeting group tumor mass show apparent red fluorescence rather than
Targeting group tumor mass does not show red fluorescence, and the fluorescence intensity absolute value of targeting group tumour is apparently higher than non-targeted group.Inject nanometer
After grain 1h, apparently higher than non-targeted group, difference has the ratio of targeting group tumor region fluorescence intensity/non-tumor region fluorescence intensity
Statistical significance (p ＜ 0.05).
3rd, tumor mass ultra-thin section
Light Microscopic observation tumor mass tissue ultra-thin section, it is seen that spread more dotted or spherical shape nanometer in the section of targeting group
Grain, and only visible a little nanoparticle in non-targeted group of section.
Cell and tumor model all select the oophoroma SKOV3 cells rich in folacin receptor.It can be seen that targeting
In experiment, targeting group SKOV3 cell peripherals and the visible more nanoparticle aggregation of cell interior, and non-targeted group of cell peripheral is basic
No nanoparticle aggregation, has absolutely proved that folic acid possesses the ability for efficiently connecting its receptor.Antagonism group is dissociated in addition after folic acid,
The folacin receptor of SKOV3 cells is occupied by free leaf, has blocked the combination of nanoparticle and cell, so cell peripheral has no apparent
Nanoparticle fluorescence is assembled, and the specificity of folic acid and cell combination is also reflected from side.In internal targeting experiment, targeting group is naked
The visible red fluorescence region for representing targeted nano granule of mouse tumor by local, and fluorescence intensity and scope became with the time
Change, and apparent fluorescence is had no always at non-targeted group of nude mice its tumour, this illustrates folate-targeted nanoparticle in live body to being rich in
The SKOV3 tumours of folic acid have good targeting.The nanoparticle grain size 300nm-400nm that the present invention makes, and tumour capillary
Vascular endothelial gap about 100nm-780nm, so nanoparticle can pass through capillary endothelium gap and reach outside blood vessel, i.e.,
EPR (Enhanced Permeation and Retention effect) effect.In targeting group and non-targeted group, nanoparticle is equal
Can near tumor cells (passive target) be reached through tumor vascular endothelium gap by EPR effects, and in targeting group, nanoparticle
Except passive target, simultaneously and then can also be swallowed with cell surface and active combination by the targeting of folic acid, i.e. active target
To.This is it is demonstrated experimentally that the nanoparticle quantity for depositing near tumor cells by active targeting and passive target two ways
Far more than the nanoparticle deposited through passive target merely.Experimental result shows, 0.5 it is small when tumour at extent of fluorescence it is smaller, 1 is small
When after the intensity of fluorescence and scope are maximum at tumour, illustrate 1 it is small when the nanoparticle that deposits of tumor by local far more than 0.5 it is small when.And
To 6 it is small when, with the reduction of the nanoparticle in blood circulation and the continuous metabolic exhaustion of tumor by local nanoparticle, cause local glimmering
Luminous intensity and scope constantly reduce.
4th, FA/Fe3O4/ PFP/HCPT@Lipid contrast agent enhancing ultrasound, optoacoustic and Magnetic Resonance Imaging research
1)FA/Fe3O4The external thermal induced phase transition of/PFP/HCPT@Lipid contrast agent and enhancing ultrasonic development research
(1) the FA/Fe3O4/PFP/HCPT@Lipid nano-emulsions of preparation are used into refrigerated centrifuge centrifugation (temperature：
4 °C, rotating speed：6000rpm, time：5min), nanoparticle is resuspended with de aerated water, prepares nanoemulsions.
(2) 2mL nanoemulsions is taken to be packed into emgloves finger tip, using the de aerated water of equivalent be packed into emgloves finger tip as pair
According to.
(3) above-mentioned emgloves is put into the water-bath for filling de aerated water, the temperature of water-bath be respectively set to 40 DEG C,
45 DEG C, 50 DEG C and 55 DEG C.
(4) Bai Sheng diasonograph (frequency probe is used：12MHz,MI:0.06) to the liquid of above-mentioned gloves finger tip in adding
It is hot front and rear to carry out ultrasonoscopy, observed using grayscale mode and imaging mode, and with DFY softwares to image echo intensity into
The difference of echo intensity is compared in row measurement.
(5) lotion after taking 10 μ L heated respectively is placed under light microscopic, observes nanoparticle form size variation.
Testing result is：
Under gray scale ultrasound pattern, when water-bath pot temperature is 40 DEG C, in uniform dotted low echo in water pocket, with heating water bath
It is before identical.When temperature reaches 45 DEG C and 50 DEG C, the visible more strong echo of bubble, bright to bubble strong echo at 55 DEG C in water pocket
It is aobvious to reduce.Under harmonic ultrasonic pattern, when water-bath pot temperature is 40 DEG C, without enhancing in water pocket.At 45 DEG C and 50 DEG C, development in water pocket
It is remarkably reinforced.Echoenhance is opposite during to 55 DEG C, in water pocket weakens.It can under light microscopic in the lotion that 45 DEG C and 50 DEG C of water pockets suction out
More bubble is seen to generate.In experimentation, intracapsular de aerated water as control is always echoless in both modes.DFY
Quantitative analysis software measurement result shows, the lotion echo intensity value highest in 50 DEG C of water pockets.
2)FA/Fe3O4The external sound of/PFP/HCPT@Lipid contrast agent causes phase transformation and enhancing ultrasonic development research
(1) 3% jellium model is made with de aerated water and gel powder, includes the hole of capacity about 2mL.
(2) 2mLFA/Fe is taken3O4/ PFP/HCPT@Lipid nanoemulsions are packed into jellium model hole, are visited using LIFU instrument
Head irradiates lotion in jellium model, and model surface coating ultrasonic coupling agent makes LIFU focuses be located in lotion.LIFU instrument
Pulse mode is arranged to, intensity is respectively 0.8W/cm2、1.6W/cm2、2.4W/cm2、3.2W/cm2, the time be respectively 1min,
2min、3min、4min.LIFU irradiation finishes, and takes a little nano-emulsion row optical microphotograph sem observation nanoparticle size variation.
(3) LIFU effects finish, and use Bai Sheng diasonograph (frequency probe：12MHz,MI:0.06) to jellium model
Interior lotion carries out ultrasonoscopy, is observed using grayscale mode and imaging mode, and with DFY softwares to image echo intensity into
The difference of echo intensity is compared in row measurement.
Testing result is：
LIFU intensity is 0.8W/cm2When, be showed no in each time point under ultrasound grayscale and imaging mode be remarkably reinforced it is aobvious
Shadow.When LIFU intensity is 1.6W/cm2When, in visible fainter enhancement effect of each time point under ultrasound grayscale and imaging mode.
When LIFU intensity is increased to 2.4W/cm2When, in visible obvious enhancing effect of each time point under ultrasound grayscale and imaging mode
It should.The discovery of DFY analysis softwares measured value, 2.4W/cm2, the echo intensity value of 2 minutes is apparently higher than 2.4W/cm2, 1 minute, difference has
Statistical significance, and and 2.4W/cm2, 3 minutes and 2.4W/cm2, 4 minutes compare, echo intensity value difference is not not statistically significant.
When LIFU intensity is increased to 3.2W/cm2When, the enhancing intensity of different time points is compared with 2.4W/cm2Decline instead.Varying strength and when
Between after LIFU irradiation the ultrasonic echo intensity value of solution see Tables 1 and 2.Intensity is 0.8W/cm2When, phase transformation is had no under microscope
Increased bubble；Intensity is 1.6W/cm2When, microscope visible bubble in bubble generates；Intensity is 2.4W/cm2When, microscope bubble increases
It is more；Intensity is 3.2W/cm2When, microscope is shown in that bubble increases, and quantity is reduced.
The gray scale ultrasound echo intensity value of solution after 1 varying strength of table and time LIFU irradiation
● with 2.4W/cm2, compared with 1 minute, p ＜ 0.05
▲ with 2.4W/cm2 .p ＞ 0.05 compared with 3 minutes
With 2.4W/cm2, compared with 4 minutes, p ＞ 0.05
The ultrasonic contrast echo intensity value of solution after 2 varying strength of table and time LIFU irradiation
With 2.4W/cm2, compared with 1 minute, p ＜ 0.05
▲ with 2.4W/cm2, compared with 3 minutes, p ＞ 0.05
With 2.4W/cm2, compared with 4 minutes, p ＞ 0.05
3)FA/Fe3O4Sound causes phase transformation and enhancing ultrasonic development research in/PFP/HCPT Lipid contrast agent bodies
(1) folate-targeted contrast agent FA/Fe3O4/PFP/HCPT@Lipid (modified with folic acid) and non-targeted contrast agent are prepared
Fe3O4/PFP/HCPT@Lipid (no modified with folic acid).
(2) tumor bearing nude mice (diameter of tumor about 1cm) is randomly divided into three groups and carries out different disposal：Group I：Tail vein injection
The parallel LIFU irradiation tumour of folate-targeted contrast agent (FA/Fe3O4/PFP/HCPT@Lipid)；Group II：Tail vein injection is non-targeted
The parallel LIFU irradiation tumour of contrast agent (Fe3O4/PFP/HCPT@Lipid)；Group III：Tail vein injection folate-targeted contrast agent
(FA/Fe3O4/PFP/HCPT@Lipid)。
(3) with 150 μ L yellow Jackets (concentration：1%) after anaesthetizing nude mice, three groups of nude mices note contrast agent about through tail vein
200μL(1mg/mL).Group I and group II nude mice in inject contrast agent 1 it is small when after row LIFU irradiate, coating couplant ensure LIFU coke
Point is located in knurl.LIFU instrument is arranged to pulse mode, and intensity is respectively 0.8W/cm2,1.6W/cm2,2.4W/cm2,3.2W/
Cm2, time are respectively 1min, 2min, 3min, 4min.
(4) LIFU effects finish, and use Bai Sheng diasonograph (frequency probe：12MHz,MI:0.06) tumour is carried out
Ultrasonoscopy is observed using grayscale mode and imaging mode, and image echo intensity is measured with DFY softwares, than
Compared with the difference of echo intensity.
Testing result is：
Before injecting contrast agent, each group nude mouse tumor gray scale ultrasound is shown as uniform low echo, no obvious liquefaction necrosis.Group
I (targeted contrast agent+LIFU groups) nude mouse tumor is in 0.8W/cm2、1.6W/cm2And 2.4W/cm2After LIFU irradiation, Ultrasonographic
It has no and is remarkably reinforced, DFY institutes survey time sound intensity angle value is shown, only in 2.4W/cm2, 3 minutes and 2.4W/cm2, after irradiation in 4 minutes, return
Sound intensity angle value increased.LIFU intensity is increased to 3.2W/cm2When, Ultrasonographic may occur in which macroscopic enhancing.
DFY software measured values show, 3.2W/cm2, the echo intensity value of 2 minutes is compared with 3.2W/cm2, apparent increase in 1 minute, difference has statistics
Learn meaning (p ＜ 0.05), 3.2W/cm2, 2 minutes echo intensity value and 3.2W/cm2, 3 minutes and 3.2W/cm2, 4 minutes ratios
Compared with no significant difference (p ＞ 0.05).And organize III (targeted contrast agent group) of II (non-targeted contrast agent+LIFU groups) and group
Gray scale ultrasound and harmonic ultrasonic have no significant change, DFY softwares measured value analysis echo intensity before and after nude mouse tumor injection contrast agent
Value is also without substantially changeing.Echo intensity value of I nude mouse tumor of group after varying strength and different time LIFU irradiation is shown in Table 3 (ashes
Rank ultrasonic echo intensity value) and table 4 (ultrasonic contrast echo intensity value).
The gray scale ultrasound echo intensity value (targeting group) after I tumours is organized after 3 varying strength of table and time LIFU irradiation
● with 3.2W/cm2, compared with 1 minute, p ＜ 0.05
▲ with 3.2W/cm2, compared with 3 minutes, p ＞ 0.05
With 3.2W/cm2, compared with 4 minutes, p ＞ 0.05
The ultrasonic contrast echo intensity value (targeting group) after I tumours is organized after 4 varying strength of table and time LIFU irradiation
● with 3.2W/cm2, compared with 1 minute, p ＜ 0.05
▲ with 3.2W/cm2, compared with 3 minutes, p ＞ 0.05
With 3.2W/cm2, compared with 4 minutes, p ＞ 0.05
Sound is caused in Phase Change Experiment in vitro, when intensity is less than 2.4W/cm2When, LIFU instrument can not promote PFP nanoparticle phases
Become.When LIFU parameters are 2.4W/cm2At 2 minutes, naked eyes see enhancing gray scale ultrasound and enhancing ultrasonic contrast is more apparent, DFY
Software, which also shows echo intensity value at this time, more significant raising, and when the time being deferred to 3 minutes and 4 minutes, naked eyes, which are seen, to be increased
Strong degree without significant difference, DFY software measured values at 2 minutes with also showing 2.4W/cm2, 2 minutes, 3 minutes, the sound intensity of 4 minutes
There was no significant difference for value.Therefore subsequent external sound cause phase transition parameter be set to 2.4W/cm2,2 minutes.Internal sound causes phase transformation
Experiment display, when LIFU parameters to 3.2W/cm2, 2 minutes when, the apparent ultrasound enhancing development of naked eyes could occur.DFY
Measured value shows, 3.2W/cm2, 2 minutes, 3 minutes, there was no significant difference for the sound intensity value of 4 minutes.Exposure time is shorter, ultrasonic energy
Will be smaller to the damage of tissue, therefore subsequent external sound causes phase transition parameter to be set to 3.2W/cm2, 2 minutes.For similary
The PFP nanoparticles of making, internal phase transformation need 3.2W/cm2, 2 minutes, and only need 2.4W/cm in vitro2, 2 minutes, reason may be
Deposit to that extravascular nanoparticle grain size is smaller by EPR effects in vivo, grain size it is big can not distributed by capillarity wall and only
It can be cycled in blood pool.The size of nanoparticle grain size causes the threshold value of phase transformation to be inversely proportional with sound, and grain size is about small, and phase transformation threshold value is higher,
Also more it is difficult to phase transformation.Group II nude mouse tumor ultrasonograms, which are showed no, to be remarkably reinforced, and is not have modified with folic acid because of nanoparticle, thus
It is seldom to gather the nanoparticle of near tumor cells, is not enough to generate phase transformation and enhance the effect of ultrasonic development.Group III nude mices are swollen
Knurl ultrasonogram, which also has no, to be remarkably reinforced, and is because lacking the excitation of ultrasonic (LIFU).This is also LIFU in group I from side illustration
Irradiation promotes phase transformation rather than nude mice body temperature promotes phase transformation.
It absolutely proves that the PFP nanoparticles prepared by the present invention have good phase-change characteristic, and can effectively increase after the phase change
Strong ultrasonic development.
5th, FA/Fe3O4/ PFP/HCPT@Lipid contrast agent inside and outside enhancing optoacoustic imaging experiment
1)FA/Fe3O4/ PFP/HCPT@Lipid contrast agent enhances photoacoustic imaging research in vitro
①Fe3O4Optoacoustic Characteristics Detection
As shown in figure 3, Fe3O4、FA/Fe3O4Tri-/PFP/HCPT@Lipid, FA/PFP/HCPT@Lipid samples with
The near-infrared laser excitation of 680nm-950nm full band ranges, Fe3O4、FA/Fe3O4The optoacoustic peak value of/PFP/HCPT@Lipid
Respectively 2.66 and 1.63, the two plots changes are consistent, and the peak value of excitation is at 705nm, and control group FA/PFP/
HCPT@Lipid microballoon photoacoustic signal peak values are only 0.37, and waveform is irregular, without special laser excitation wavelength scope.
2. various concentration Fe3O4Photoacoustic signal detection
Under optoacoustic instrument laser excitation, the photoacoustic signal of gel module is with Fe3O4The rise of concentration and gradually enhance；No
Containing Fe3O4Control group do not occur apparent photoacoustic signal.The optoacoustic value of each group such as table 5.
5 various concentration Fe of table3O4Photoacoustic signal value
* compare with control group, p ＜ 0.05
3. cell opto-acoustic result
FA/Fe3O4After/PFP/HCPT@Lipid nanoparticles are combined or swallowed by SKOV3 cell-targetings, photoacoustic imaging is shown
Uniform photoacoustic signal, with the extension of nanoparticle and SKOV3 cell incubation times, photoacoustic signal substantially increases.And control group
Cell suspension naked eyes, which are seen, to be creamy white, and has no apparent optoacoustic imaging.Optoacoustic value quantitative analysis, as nanoparticle is thin with SKOV3
The extension of born of the same parents' incubation time, optoacoustic value substantially increase.
2)FA/Fe3O4Enhancing photoacoustic imaging experiment in/PFP/HCPT Lipid contrast agent body
Before injecting contrast agent, each group nude mouse tumor is showed no apparent photoacoustic signal.Group I nude mice injection folate-targeted radiography
After agent 0.5h, the visible photoacoustic signal of tumor by local, and being remarkably reinforced in 1h photoacoustic signals, photoacoustic signal gradually weakens during 6h.And
After group II and group III nude mices injection contrast agent, in being showed no apparent photoacoustic signal in three time points.The optoacoustic quantitative values of each group
Such as table 6.
Table 6 is injected before contrast agent and 0.5h afterwards, 1h and 6h tumor by local photoacoustic signal values
Compare with before injection, p ＜ 0.05
★ and group II compare, p ＜ 0.05
● and group III compares, p ＜ 0.05
In enhancing in vitro in photoacoustic imaging research, FA/Fe is detected3O4/ PFP/HCPT@Lipid contrast agent and Fe3O4
Solution is the same characteristic absworption peak at 705nm, and waveform traveling is consistent.Without the sub- Fe of light absorption3O4Gel piece optoacoustic
Pickup electrode is weak, and is mixed with FA/Fe3O4The gel of/PFP/HCPT@Lipid contrast agent shows stronger optoacoustic Contrast enhanced and makees
With, and with FA/Fe3O4The increase of/PFP/HCPT@Lipid contrast medium concentrations, photoacoustic signal are remarkably reinforced, and optoacoustic value is also apparent
Rise, difference are statistically significant.FA/Fe3O4After/PFP/HCPT@Lipid contrast agent and SKOV3 cell incubations, cell suspension
Photoacoustic signal can be generated, and as the extension of common incubation time, photoacoustic signal are also remarkably reinforced, illustrate to be combined by cell-targeting and
After phagocytosis, Fe3O4Keep its optoacoustic enhancement effect.Confirm the FA/Fe304/PFP/HCPT@Lipid contrast agent that the present invention is developed
With the potential as photoacoustic imaging contrast-enhancing agent.
Enhance in vivo in photoacoustic imaging research, contain Fe through nude mice tail vein injection3O4Targeting contrast agent FA/
Fe3O4/ PFP/HCPT@Lipid, are assembled by active targeting and passive target, and tumor by local contains the nanoparticle of higher concentration
FA/Fe3O4/ PFP/HCPT Lipid, improve the sensitivity of local photoacoustic signal.Three time points of Germicidal efficacy：0.5h,
1h and 6h.It is weaker in the photoacoustic signal that 0.5h tumor by local detects, it may be possible to which that the nanoparticle for gathering tumor by local is less.And
When 1h, as the nanoparticle in cycling constantly deposits to the active targeting of tumor by local and folic acid by EPR effects
Effect, the nanoparticle of tumor by local deposition gradually increases, therefore detects stronger photoacoustic signal.To the 6th it is small when, in cycling
The continuous reduction of nanoparticle and the continuous metabolic exhaustion of tumor by local nanoparticle so that tumor locus nanoparticle concentration by
Decrescence few, the optoacoustic enhancing signal of generation also gradually weakens.
The contrast agent FA/Fe of the present invention3O4/ PFP/HCPT@Lipid can enhance photoacoustic imaging in inside and outside, to realize
Experiment basis are established in the multi-modal imagings of FA/Fe304/PFP/HCPT@Lipid.
6th, FA/Fe3O4/ PFP/HCPT@Lipid contrast agent inside and outside enhances Magnetic Resonance Imaging experimental study
1)FA/Fe3O4/ PFP/HCPT@Lipid contrast agent enhances MRI investigation in vitro
(1) preparation of experimental group：FA/Fe304/PFP/HCPT@Lipid nano-emulsions are diluted to 1% gel different
Concentration is as experimental group：III：5μg mL-1, IV：10μg mL-1, V：20μg mL-1, VI：40μg mL-1, VII：80μg mL-1,
VIII：160μg mL-1, IX：320μg mL-1, X：640μg mL-1, XI：1280μg mL-1, XII：2560μg mL-1.Take degassing
Water and 1% gel without nanoparticle I and control Group II as a control group.
2) result is：As shown in figure 4, FA/Fe3O4/ PFP/HCPT@Lipid develop in T2*WI imagings in negativity enhancing,
Compared with de aerated water, without Fe3O4Jellium model have no clear signal strength reduction (P ＞ 0.05), and FA/ containing various concentration
Fe3O4There is different degrees of reduction (P ＜ 0.05) in the signal strength of each group (III-XII) of/PFP/HCPT@Lipid, and
And with the increase of concentration, MR signal strengths are gradually reduced.When concentration is up to 640 μ g mL-1(corresponding Fe concentration is 53.3 μ g
mL-1) when, signal strength declines most apparent in EP pipes, almost in shadow, but when concentration is more than 640 μ g mL-1, the magnetic in EP pipes
Geometry deformation has occurred in resonance image.Table 7 is de aerated water, gel and various concentration FA/Fe3O4/ PFP/HCPT@Lipid solution institute
Corresponding Fe concentration and magnetic resonance signal intensity value.
The FA/Fe of 7 various concentration of table3O4The concentration of iron and magnetic resonance signal intensity of/PFP/HCPT@Lipid solution compare
▲ compared with organizing I .P ＜ 0.05
* the .P ＜ 0.05 compared with group II
2)FA/Fe3O4Enhancing MRI investigation in/PFP/HCPT Lipid contrast agent body
Compared with before injection contrast agent, there is negativity enhancing after group I nude mice injection targeted contrast agent, and after injection
1h is more apparent, and magnetic resonance signal intensity is significantly lower than (p before injection after 1h and 6h after injection contrast agent<0.05).Group II and group III
After nude mice injection contrast agent, apparent magnetic resonance negativity enhancing is showed no, magnetic resonance signal intensity is also without significant change (p>0.05).
In vitro in Magnetic Resonance Imaging experiment, control group (de aerated water and without Fe3O4Gel) in high RST, and be mixed with
FA/Fe3O4The experimental group of/PFP/HCPT@Lipid nanoparticles is in low signal, with continuous rise, image gradually becomes dim,
T2 signal strength values continuously decrease, and absolutely prove FA/Fe3O4The magnetic-particle Fe loaded in/PFP/HCPT@Lipid shell membranes3O4
It is the reason for it enhances the development of magnetic resonance negativity place.In vivo in Magnetic Resonance Imaging experiment, by tail vein injection contrast agent,
Evaluate Fe3O4To magnetic resonance contrast's humidification of nude mouse tumor.It turns out that injection targeted contrast agent FA/Fe3O4/PFP/
After HCPT@Lipid, there is different degrees of negativity enhancing development in nude mouse tumor magnetic resonance, and control group (injects non-targeted radiography
Agent and without Fe3O4Contrast agent) within observing time tumor tissues magnetic resonance signal intensity without substantially changeing.In addition, it is noting
Penetrate targeted contrast agent FA/Fe3O4The enhancing of 0.5h magnetic resonance negativity is weaker after/PFP/HCPT@Lipid, and 1h is stronger, because in 1h,
It is more that the nanoparticle of tumor by local is deposited to by active targeting (folate-targeted) and passive target (EPR effects), local Fe3O4
Concentration is higher.
It can be seen that the targeted contrast agent FA/Fe of the present invention3O4/ PFP/HCPT@Lipid, being capable of negativity in inside and outside
Enhance magnetic resonance imaging.
6th, FA/Fe3O4/ PFP/HCPT@Lipid contrast agent targeted therapies oophoroma is tested
1)FA/Fe3O4/ PFP/HCPT@Lipid contrast agent and drug distribution
HCPT is the drug with strong anti-tumor effect extracted from China endemic plant camplotheca acuminata, is that DNA topologys are different
Structure enzyme I inhibitor is widely used in the treatment of the Several Kinds of Malignancy such as liver cancer, stomach cancer, leukaemia, but the not fat melting of HCPT water is difficult
Molten, circulation time in vivo is short, lactonic ring structural instability so that its clinical practice is restricted, and the present invention arrives HCPT packages
In nanoparticle lipid shell, increase its dissolubility, extend circulation time.Nanoparticle is acted on by active and passive target, deposition
To near tumor cells, after LIFU excitations, liquid fluorocarbon occurs liquid-gas phase transition and discharges drug in nanoparticle, so as to dramatically increase
Tumor by local drug concentration.
As a result it is：
①FA/Fe3O4Distribution in/PFP/HCPT Lipid nanoparticles body
Under inverted fluorescence microscope, non-targeted group of nude mouse tumor ultra-thin section each time point is showed no apparent red glimmering
Light.And targeting group nude mouse tumor ultra-thin section, in 0.5h, 1h, 6h, 12h, equal red color visible fluorescence for 24 hours, fluorescence is most bright during 1h
It is aobvious.
2. nude mice blood drug concentration is distributed
HCPT parenteral solutions group and FA/Fe3O4The blood concentration of/PFP/HCPT@Lipid nano-emulsion group nude mices is shown quickly
Downward trend, but in each detection time point, FA/Fe3O4The blood concentration of/PFP/HCPT@Lipid nano-emulsion group nude mices
It is above HCPT parenteral solution groups.After injection 6 it is small when, HCPT parenteral solution groups are substantially not detectable blood concentration, and FA/Fe3O4/
PFP/HCPT@Lipid nano-emulsion groups still can detect that up to 198ng mL after 24 hours-1Blood concentration.
3. nude mouse tissue drug concentration is distributed
Due to the package of lipidic shell, FA/Fe3O4The retention time of HCPT in vivo is compared with HCPT in/PFP/HCPT@Lipid
Parenteral solution significantly extends, even if after when injection 24 is small, the presence of drug HCPT can be still detected in each tissue, and is injected in HCPT
Group, 12 can't detect drug when small.Especially for tumor tissues, FA/Fe3O4/ PFP/HCPT@Lipid injection group drug concentrations
It is significantly higher than HCPT injection groups, and the drug retention time is obviously prolonged.
4. tumor tissue drug concentration distribution after LIFU effects
FA/Fe3O4The tumor tissue drug concentration of/PFP/HCPT@Lipid nanoemulsion injection groups is significantly higher than HCPT notes
Penetrate group (P<0.05), FA/Fe3O4The tumor tissue drug concentration of/PFP/HCPT@Lipid+LIFU groups is significantly higher than FA/
Fe3O4/ PFP/HCPT@Lipid nanoemulsion injection groups (P<0.05).
Spleen largely swallows FA/Fe in this experiment3O4/ PFP/HCPT@Lipid nanoparticles are considered as that nanoparticle has length
The evidence of cycle characteristics.In addition, although liver has very high HCPT drug concentrations, the HCPT of routine dose can only influence stomach
Intestines and medulla hematopoietic system, without liver injury.Drug concentration is equal at each detection time point, the tumour of HCPT injection groups
Less than FA/Fe3O4/ PFP/HCPT@Lipid nanoparticle injection groups.After injection 6 it is small when, examined at the tumour of HCPT injection groups
Drug concentration is not detected, and in FA/Fe3O4In the tumor tissues of/PFP/HCPT@Lipid nanoparticle injection groups, 24 after injection
Hour remains to detect 50ng g-1Drug concentration.The FA/Fe of modified with folic acid3O4/ PFP/HCPT@Lipid can improve tumour
Local Tissue, and longer time can be kept, the killing tumor cell long lasting for property will be played the role of.
In addition, retain in liver, spleen, lung, nephrocardiac nanoparticle, by the continual HCPT to blood pool release package,
It is beneficial to keep higher blood levels.After LIFU effects, the FA/Fe of tumor by local aggregation is promoted3O4/PFP/
HCPT@Lipid phase transformations simultaneously discharge drug, so as to improve local drug concentration, enhance drug fragmentation effect.
2)FA/Fe3O4/ PFP/HCPT@Lipid contrast agent is to the targeted therapy effect of oophoroma
Nude mice SKOV3 tumor inoculation situations：After inoculated tumour cell, nude mice feed, activity and the state of mind are normal,
Tumour growth is good.The nude mice 30 for choosing diameter of tumor about 1mm is only tested.
As a result it is：
1. the therapeutic effect of tumour
(1) growth index is as shown in figure 5, FA/Fe3O4/ PFP/HCPT@Lipid+LIFU group nude mouse tumors growth index is most
It is low.HCPT groups and FA/Fe3O4The growth index of/PFP/HCPT@Lipid groups is without significant difference.FA/Fe3O4/PFP@Lipid+
LIFU groups and Fe3O4Effect of/PFP/HCPT@Lipid the groups without apparent control tumour growth.
(2) the 14th day after the treatment takes off neck and puts to death nude mice, completely takes out tumour, and experimental group nude mouse tumor is less than brine
Control group, FA/Fe3O4The tumour of/PFP/HCPT@Lipid+LIFU group nude mices is minimum.
(3) as shown in table 8, FA/Fe3O4/ PFP/HCPT@Lipid+LIFU groups show highest tumour inhibiting rate
14th day tumor quality and tumour inhibiting rate (mean ± standard deviation, n=5) after table 8 is treated
* P ＜ 0.01vs control group
△ P ＜ 0.01vs FA/Fe3O4PFP/HCPT@Lipid group
2. immunohistochemical detection result
(1) cell Proliferation and proliferation index：PCNA is the results show that six groups of nude mouse tumors equal visible cell core under microscope
Interior yellow particle calculates positive rate so as to obtain proliferation index PI, the results show FA/Fe3O4/ PFP/HCPT@Lipid+LIFU groups
Proliferation index it is minimum (#p ＜ 0.05), control group (physiological saline group) proliferation index highest.
(2) Apoptosis and apoptotic index：TUNEL is the results show that each group tumour visible brown color under microscope
Grain calculates positive rate and obtains apoptotic index AI, the results show FA/Fe3O4The apoptotic index of/PFP/HCPT@Lipid+LIFU groups is most
High (* p ＜ 0.05), control group apoptotic index is minimum.
In this experiment, FA/Fe3O4/ PFP/HCPT@Lipid+LIFU group tumour growths are most slow, and volume is minimum, immunohistochemistry
Show that this group of tumor cell proliferation is minimum and apoptosis is most.FA/Fe3O4/ PFP/HCPT@Lipid groups are because of the work without LIFU
With nanoparticle is weaker to the inhibitory action of tumour.HCPT groups are the equal of a kind of Formulations for systemic administration, and the medication amount of tumor by local is few,
So therapeutic effect is unsatisfied with.FA/Fe3O4/ PFP@Lipid+LIFU groups have LIFU to act on and participated in without drug, the suppression to tumour
It makes of weak, from side illustration, the nanoparticle of packaging medicine combines importance of the LIFU for tumor-killing.Fe3O4/PFP/
HCPT@Lipid groups lack folic acid volume targeting, also lack the excitation of LIFU, to tumour substantially without obvious therapeutic action.
The nanoparticle FA/Fe of modified with folic acid3O4/ PFP/HCPT Lipid are reached by active and passive target effect and are incorporated into ovary
Carcinoma cells generate a series of physical chemical action by the excitation of LIFU, so as to generate therapeutic effect to tumour cell.
The introducing of LIFU in this problem, at least effect there are three aspect.The PFP liquid-gas phase transitions of first, LIFU induction, which can cause, to be received
Grain of rice FA/Fe3O4/ PFP/HCPT@Lipid discharge drug HCPT, and drug that is direct and being obviously improved tumor by local is dense
Degree.Many scholars also demonstrate that ultrasound can promote microballoon or microvesicle to discharge the drug included in it.Second, it is formed after phase transformation
Gas-filled microbubble under the action of ultrasound, can generate a series of physical effect, such as acoustic radiation force (acoustic
Radiation forces) and cavitation effect (acoustic cavitation), microjet (jets of can be generated
Fluid), so as to transient destruction tumour cell wall, intake of the tumour cell to drug is increased.3rd, microjet is simultaneously
Transient destruction tumour capillary endothelial cell gap, nanoparticle and drug in blood pool can penetrate into tissue space,
So as to generate the fragmentation effect of bigger to tumour cell.
Therefore, the nanoparticle FA/Fe that the present invention develops3O4/ PFP/HCPT@Lipid cooperation low-strength focusing ultrasonics LIFU's
Effect, has good therapeutic effect to ovarian Cancer of Nude Mice.
Claims (10)
1. the multi-modal imaging nano-contrast agent of inversion of phases is targeted, including lipid shell membrane, it is characterised in that：It is mounted on lipid shell membrane
Oleic acid oxidation iron nano-particle and drug HCPT, and it is modified with folic acid on lipid shell membrane.
2. the targeting multi-modal imaging nano-contrast agent of inversion of phases according to claim 1, it is characterised in that：The lipid
Liquid fluorocarbon is enclosed with inside shell membrane.
3. the targeting multi-modal imaging nano-contrast agent of inversion of phases according to claim 2, it is characterised in that：Its grain size is
321 ± 67nm, grain size polydispersity index PDI are 0.43.
4. the targeting multi-modal imaging nano-contrast agent of inversion of phases according to claim 3, it is characterised in that：Its ZETA current potential
For -50mV.
5. the targeting multi-modal imaging nano-contrast agent of inversion of phases according to claim 4, it is characterised in that：Its HCPT is encapsulated
Rate is 60.51% ± 2.33%, and drugloading rate is 8.33% ± 0.57%.
6. target the preparation method of the multi-modal imaging nano-contrast agent of inversion of phases, it is characterised in that：Using rotary evaporation-ultrasound
Method.
7. the preparation method of the multi-modal imaging nano-contrast agent of targeting inversion of phases according to claim 6, it is characterised in that：
The operating procedure of rotary evaporation-ultrasonic method is as follows：
1), dissolve：First by oleic acid oxidation iron nano-particle, drug HCPT and the lipid shell for including phosphatide-folic acid synthesis material
After film raw material is dissolved with organic solvent；
2), rotary evaporation：Decompression rotary evaporation, which is carried out, in Rotary Evaporators removes organic solvent；
3), it is cleaned by ultrasonic：It is placed in again in ultrasonic cleaning agent and cleans vibration；
4), centrifuge：4 DEG C of refrigerators of postposition are centrifuged, obtain sample.
8. the preparation method of the multi-modal imaging nano-contrast agent of targeting inversion of phases according to claim 7, it is characterised in that：
After the ultrasonic cleaning of step 3), further included step 3a), add in liquid fluorocarbon, after being emulsified using sound and vibration instrument, then will emulsification
Liquid carries out the operation of step 4).
9. the preparation method of the multi-modal imaging nano-contrast agent of targeting inversion of phases according to claim 8, it is characterised in that：
Solution temperature in the step 1) is 50 DEG C；
The rotating speed of Rotary Evaporators in the step 2) is rotating speed：80rpm, when the time is 2 small；
The step 3a) in the whole ice bath of sound and vibration instrument emulsification, and power is 100w, time 6min；
The temperature centrifuged in the step 4) is 4 DEG C, rotating speed 6000rpm, time 5min, is centrifuged altogether three times.
10. the preparation method of the multi-modal imaging nano-contrast agent of targeting inversion of phases according to claim 9, feature exist
In：Step 3a) in sound and vibration instrument by the way of sound and vibration is interrupted.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109966514A (en) * | 2019-04-26 | 2019-07-05 | 湖北科技学院 | A kind of phase transition targeted nano bubble, preparation method and application |
| CN111110657A (en) * | 2020-02-14 | 2020-05-08 | 徐州医科大学 | Nano microsphere capable of realizing dual-mode imaging and targeted therapy of breast cancer and preparation method thereof |
| CN111557926A (en) * | 2020-05-09 | 2020-08-21 | 重庆医科大学 | Targeting phase-change nano-drug system and preparation method and application thereof |
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2017
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109966514A (en) * | 2019-04-26 | 2019-07-05 | 湖北科技学院 | A kind of phase transition targeted nano bubble, preparation method and application |
| CN111110657A (en) * | 2020-02-14 | 2020-05-08 | 徐州医科大学 | Nano microsphere capable of realizing dual-mode imaging and targeted therapy of breast cancer and preparation method thereof |
| CN111557926A (en) * | 2020-05-09 | 2020-08-21 | 重庆医科大学 | Targeting phase-change nano-drug system and preparation method and application thereof |
| CN111557926B (en) * | 2020-05-09 | 2021-12-03 | 重庆医科大学 | Targeting phase-change nano-drug system and preparation method and application thereof |
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