CN109481701A - Glioma image nano-probe and its preparation method and application - Google Patents
Glioma image nano-probe and its preparation method and application Download PDFInfo
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- CN109481701A CN109481701A CN201711440558.1A CN201711440558A CN109481701A CN 109481701 A CN109481701 A CN 109481701A CN 201711440558 A CN201711440558 A CN 201711440558A CN 109481701 A CN109481701 A CN 109481701A
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
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Abstract
The present invention provides a kind of glioma image nano-probes and its preparation method and application, it is related to nano-probe technical field, the glioma image nano-probe is mainly by carboxylated modified liposomes, contrast agent and lactoferrin are prepared, the contrast agent contains in the carboxylated modified liposomes, the lactoferrin and the carboxylated modified liposomes pass through chemistry key connection, alleviate existing glioma obscure boundary, operation is difficult to the technical issues of thoroughly cutting off, glioma image nano-probe provided by the invention not only biodegradable, good water solubility, and blood-brain barrier can be passed through, active targeting brain glioblastoma cell, the imaging of tumour is realized in the microenvironment of glioma, to accurately identify the boundary of glioma, improve the diagnostic accuracy of glioma, it is mentioned for human glioma operation excision For reference, it helps to cut off brain colloid completely, reduces the technical effect of the generation of sequelae.
Description
Technical field
The present invention relates to nano-probe technical fields, more particularly, to a kind of glioma image nano-probe and its preparation
Methods and applications.
Background technique
Glioma is a kind of intracranial malignant tumor from Deiter's cells and a kind of Exemplary gene saltant type
Central nervous system diseases, account for about the 40-50% of intracranial tumour.The World Health Organization (WHO) is according to the pernicious journey of glioma
Degree is classified as pilocytic astrocytoma, low potential malignancy glioma, change astrocytoma and pleomorphism glioblast
Four grades of tumor, wherein glioblastoma multiforme grade malignancy highest have a high mortality, easy to recur and refractory be cured
Feature, 2 years survival rates are still below 30% after the means treatment such as underwent operative excision, radiotherapy and chemotherapy, 5 years survival rates less than 10%,
Median survival time is only 12-15 months, it has also become the cause of death of 34-54 years old tumor patient front three.
Currently, clinical treatment glioma is based on surgical resection therapy, due to brain matter tumor and other intracranial tumours
Characteristic is not very identical, it is led in " crab foot sample " local infiltration and to the normal brain activity group infiltrative growth of primary lesion periphery
Cause tumor boundaries unclear, operation is difficult to thoroughly cut off, and causes postoperative easily to recur.Usual glioma easily occurs important in brain
Functional areas, such as cognition, movement and the language nerve center control zone of people, thus it is postoperative bring very big sequelae to patient, sternly
Ghost image rings postoperative life quality.Therefore, it is urgent to provide one kind can accurately identify the image probe on glioma boundary to improve
The recognition capability of tumor boundaries.
In view of this, the present invention is specifically proposed.
Summary of the invention
The glioma image that glioma boundary can be accurately identified one of the objects of the present invention is to provide one kind is received
Rice probe, to alleviate existing glioma obscure boundary, operation is difficult to the technical issues of thoroughly cutting off.
Glioma image nano-probe provided by the invention, including carboxylated modified liposomes, contrast agent and newborn iron egg
White, the hydrophobic layer of the carboxylated modified liposomes contains the contrast agent, the hydrophilic layer of the carboxylated modified liposomes with
For the lactoferrin by chemistry key connection, the contrast agent includes fluorescent contrast agent, photoacoustic contrast agent and fluorescence optoacoustic bimodulus
At least one of state contrast agent, preferably fluorescence optoacoustic bimodal contrast agent.
Further, the carboxylated modified liposomes are the poly ethyldiol modified liposome of carboxylated；
Preferably, in the poly ethyldiol modified liposome of carboxylated, the degree of polymerization of polyethylene glycol is 1000-10000, preferably
2000-5000。
Further, the poly ethyldiol modified liposome of the carboxylated is selected from the poly ethyldiol modified phosphatidyl gallbladder of carboxylated
The poly ethyldiol modified dipalmitoylphosphatidylcholine of alkali, carboxylated, the poly ethyldiol modified phosphatidyl-ethanolamine of carboxylated and carboxyl
At least one of modified Distearoyl Phosphatidylcholine of polyethylene glycol.
Further, the fluorescent contrast agent is selected from least one of Cy3, Cy5 or Cy5.5；
Preferably, the photoacoustic contrast agent is selected from the molybdenum disulfide or tungsten disulfide of lamella；
Preferably, the fluorescence optoacoustic bimodal contrast agent is selected from indoles cyanine dyes and/or heptamethine cyanine；
Preferably, the indoles cyanine dyes is indocyanine green；
Preferably；The seven methines cyanine dyes is selected from IR-780, IR-775, IR-797, IR-792, IR-806 or IR-
At least one of 808.
The second object of the present invention is to provide the preparation method of above-mentioned glioma image nano-probe, including walks as follows
It is rapid:
(a) carboxylated modified liposomes and contrast agent are added in organic solvent and are uniformly mixed, added in water and mix
It is even, it contains contrast agent in the hydrophobic layer of carboxylated modified liposomes, obtains liposome probe；
(b) liposome probe is uniformly mixed with carboxyl activator, adds lactoferrin and be uniformly mixed, obtains brain glue
Matter tumor image nano-probe.
Preferably, the organic solvent is volatile organic solvent, preferably in ethyl alcohol, chloroform, methylene chloride and acetone
At least one, more preferably ethyl alcohol.
Preferably, carboxyl activator is n-hydroxysuccinimide and/or 1- ethyl -3- (3- dimethyl aminopropyl)-carbon
Change diimine, preferably n-hydroxysuccinimide.
Further, the mass ratio of the carboxylated modified liposomes and the contrast agent is (1-10): 1, preferably (1-
2):1。
Further, the mass ratio of the carboxylated modified liposomes and the lactoferrin is 1:(1-3), preferably 1:
(1.5-2)。
It further, further include the purifying of liposome probe in step (a), the purifying is including organic solvent and not
Contain the removal of contrast agent；
Preferably, organic solvent is volatilized using inert gas purge and is removed；
Preferably, the contrast agent of unentrapped is removed using centrifugation.
It further, further include the purifying of glioma image nano-probe in step (b), the purifying includes carboxylic
The removal of base activator and unreacted lactoferrin；
Preferably, carboxyl activator and unreacted lactoferrin are removed using dialysis or ultrafiltration.
The third object of the present invention be to provide above-mentioned glioma image nano-probe prepare Diagnosing Gliomas and/
Or the application in treatment drug.
Glioma image nano-probe provided by the invention not only biodegradable, good water solubility, but also can pass through
Blood-brain barrier, active targeting brain glioblastoma cell realize the imaging of tumour, to accurately identify in the microenvironment of glioma
The diagnostic accuracy of glioma is improved on the boundary of glioma, provides reference for human glioma operation excision, helps brain glue
Matter excision is clean, reduces the generation of sequelae, improves postoperative life quality.
The preparation method simple process of glioma image nano-probe provided by the invention, it is easy to operate, it can be applicable in
In large-scale production.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the preparation process schematic diagram that glioma provided by the invention influences nano-probe；
Fig. 2 is that the near-infrared of the nude mice for the glioma image nano-probe solution that the injection embodiment of the present invention 2 provides is glimmering
Light figure；
Fig. 3 is the optoacoustic figure for injecting the nude mice for the glioma image nano-probe that the embodiment of the present invention 2 provides.
Specific embodiment
Technical solution of the present invention will be clearly and completely described below, it is clear that described embodiment is this hair
Bright a part of the embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not having
Every other embodiment obtained under the premise of creative work is made, shall fall within the protection scope of the present invention.
According to an aspect of the present invention, the present invention provides a kind of glioma image nano-probes, mainly by carboxyl
To change modified liposomes, contrast agent and lactoferrin to be prepared, the contrast agent contains in the carboxylated modified liposomes,
By chemistry key connection, the contrast agent includes fluorescent contrast agent, light for the lactoferrin and the carboxylated modified liposomes
At least one of sound contrast agent and fluorescence optoacoustic bimodal contrast agent, preferably fluorescence optoacoustic bimodal contrast agent.
Lactoferrin is a kind of important nonheme iron combination glycoprotein in human body, it is not only involved in the transhipment of iron, resists
Oxidation adjusts the powerful biological functions such as immune system, and it passes through blood-brain barrier function.As glioma develops, brain
Glioma surface height expresses lactoferrin receptor, therefore lactoferrin is one active targeting molecule of glioma.
Glioma image nano-probe provided by the invention is by passing through lactoferrin and carboxylated modified liposomes
Chemistry key connection, to enhance the targeting of glioma image nano-probe, can blood-brain barrier, be imaged, thus
The boundary for monitoring glioma, improves the diagnostic accuracy of glioma.
In glioma image nano-probe provided by the invention, carboxylated modified liposomes hydrophobic layer contains radiography
Agent provides protection for contrast agent, and contrast agent is delivered into glioma microenvironment, is imaged.
Glioma image nano-probe provided by the invention not only biodegradable, good water solubility, but also can pass through
Blood-brain barrier, active targeting brain glioblastoma cell realize the imaging of tumour, to accurately identify in the microenvironment of glioma
The diagnostic accuracy of glioma is improved on the boundary of glioma, provides reference for human glioma operation excision, helps brain glue
Matter excision is clean, reduces the generation of sequelae, improves postoperative life quality.
In the preferred embodiment of the present invention, carboxylated modified liposomes are the poly ethyldiol modified lipid of carboxylated
Body.
In the preferred embodiment of the invention, using the poly ethyldiol modified liposome of carboxylated, contrast agent is provided
More effectively protection, avoids being quenched for contrast agent.
In present invention further optimization embodiment, in the poly ethyldiol modified liposome of carboxylated, polyethylene glycol
The degree of polymerization is 1000-10000, preferably 2000-5000.
If the degree of polymerization of polyethylene glycol is too small, carboxylated modified liposomes cannot provide comprehensive protection for contrast agent,
If the degree of polymerization of polyethylene glycol is too big, carries out the modifies difficulty of lipid and increase, the poly ethyldiol modified liposome of carboxylated
Preparation efficiency is too low, proves through test of many times, when the degree of polymerization of polyethylene glycol is 1000-10000, carboxylated modified poly (ethylene glycol)
Liposome can provide comprehensive protection for contrast agent, especially when the degree of polymerization of polyethylene glycol is 2000-5000, the poly- second of carboxylated
Glycol modification liposome to the protecting effect of contrast agent more preferably.
In the preferred embodiment of the invention, the typical but non-limiting degree of polymerization of polyethylene glycol is 1000,
1500、2000、2500、3000、3500、4000、4500、5000、5500、6000、6500、7000、7500、8000、8500、
9000,9500 or 10000.
In present invention further optimization embodiment, the poly ethyldiol modified liposome of carboxylated is selected from the poly- second of carboxylated
Poly ethyldiol modified dipalmitoylphosphatidylcholine (the DPPC- of glycol modification phosphatidyl choline (PC-PEG-COOH), carboxylated
PEG-COOH), the poly ethyldiol modified phosphatidyl-ethanolamine of carboxylated (PE-PEG-COOH) and carboxylated poly ethyldiol modified two are hard
At least one of acyl phosphatidyl choline (DSPE-PEG-COOH).
The poly ethyldiol modified liposome of carboxylated can be the poly ethyldiol modified phosphatidyl choline of carboxylated, the poly- second of carboxylated
The poly ethyldiol modified phosphatidyl-ethanolamine of glycol modification dipalmitoylphosphatidylcholine, carboxylated or carboxylated are poly ethyldiol modified
Distearoyl Phosphatidylcholine, or above-mentioned any two kinds of mixture, such as the poly ethyldiol modified phosphatidyl gallbladder of carboxylated
Alkali and the mixture of the poly ethyldiol modified dipalmitoylphosphatidylcholine of carboxylated, the poly ethyldiol modified phosphatidyl ethanol of carboxylated
The mixture etc. of amine and the poly ethyldiol modified Distearoyl Phosphatidylcholine of carboxylated, or above-mentioned any three kinds of mixing
Object, such as the poly ethyldiol modified phosphatidyl choline of carboxylated, the poly ethyldiol modified dipalmitoylphosphatidylcholine of carboxylated and carboxyl
The mixture of polyethylene glycol modified phospholipid acyl ethanol amine, the poly ethyldiol modified dipalmitoylphosphatidylcholine of carboxylated, carboxyl
The mixture of polyethylene glycol modified phospholipid acyl ethanol amine and the poly ethyldiol modified Distearoyl Phosphatidylcholine of carboxylated, may be used also
Think above-mentioned four kinds of mixture: poly ethyldiol modified two palmityl of the poly ethyldiol modified phosphatidyl choline of carboxylated, carboxylated
The poly ethyldiol modified phosphatidyl-ethanolamine of phosphatidyl choline, carboxylated and the poly ethyldiol modified distearoylphosphatidyl gallbladder of carboxylated
The mixture of alkali.
In the preferred embodiment of the present invention, fluorescent contrast agent in Cy3, Cy5 or Cy5.5 at least one
Kind.
In the preferred embodiment of the present invention, photoacoustic contrast agent is selected from the molybdenum disulfide or tungsten disulfide of lamella.
In the preferred embodiment of the present invention, fluorescence optoacoustic bimodal contrast agent be selected from indoles cyanine dyes and/
Or heptamethine cyanine.
In present invention further optimization embodiment, indoles cyanine dyes is indocyanine green (ICG)；Seven methine cyanines
Dyestuff is selected from least one of IR-780, IR-775, IR-797, IR-792, IR-806 or IR-808, still more preferably for
Indocyanine green (ICG).
ICG is the dyestuff of a kind of near-infrared fluorescent and optoacoustic, it has in the near infrared region absorbs well, and 808nm swashs
It gives, a large amount of heat can be generated rapidly and issue photoacoustic signal, be suitble to photoacoustic imaging, ICG has transmitting close near infrared region
IR fluorescence property, therefore ICG can be used for near-infrared fluorescent/optoacoustic bimodal imaging.In addition, ICG has light sensitivity, also can
It is enough in tumour light treatment.
When selecting ICG contrast agent the most, glioma image nano-probe provided by the invention can not only be in brain colloid
Near-infrared fluorescence imaging is carried out in tumor microenvironment, and is able to carry out photoacoustic imaging, while can also carry out light to glioma
Treatment.
According to the second aspect of the invention, the present invention provides the preparation sides of above-mentioned glioma image nano-probe
Method includes the following steps:
(a) carboxylated modified liposomes and contrast agent are added in organic solvent and are uniformly mixed, added in water and mix
It is even, it contains contrast agent in modified liposomes, obtains liposome probe；
(b) liposome probe is uniformly mixed with carboxyl activator, adds lactoferrin and be uniformly mixed, obtains brain glue
Matter tumor image nano-probe.
Fig. 1 is the preparation process schematic diagram of glioma image nano-probe provided by the invention；It will be seen from figure 1 that
Glioma image nano-probe provided by the invention is made by carboxylated modified liposomes and contrast agent during mixing
Shadow agent is the inside for being coated on carboxylated modified liposomes, and then under carboxyl activator effect, carboxyl and lactoferrin are sent out
Raw reaction, so that carboxy-modified liposome and lactoferrin obtain glioma image nano-probe by chemistry key connection.
The preparation method simple process of glioma image nano-probe provided by the invention, it is easy to operate, it can be applicable in
In large-scale production.
In the preferred embodiment of the present invention, in step (a), organic solvent is volatile organic solvent, excellent
It is selected as at least one of ethyl alcohol, ethyl alcohol, chloroform, methylene chloride and acetone, more preferably ethyl alcohol.
By selecting volatile organic solvent to dissolve carboxylated modified liposomes and contrast agent, in order to subsequently through volatilization
Organic solvent is removed, organism is damaged to avoid the presence of organic solvent.
In the preferred embodiment of the present invention, carboxyl activator is n-hydroxysuccinimide (EDC) and/or 1-
Ethyl -3- (3- dimethyl aminopropyl)-carbodiimides, preferably n-hydroxysuccinimide (NHS).
In step (b), by the way that liposome probe to be uniformly mixed with carboxyl activator, to promote carboxy-modified liposome
With lactoferrin by chemistry key connection, to enhance the energy that glioma image probe provided by the invention penetrates blood-brain barrier
Power enhances its targeting to glioma.
In the preferred embodiment of the present invention, the mass ratio of carboxylated modified liposomes and contrast agent is (1-
10): 1, preferably (1-2): 1.
In the preferred embodiment of the invention, the typical but non-limiting matter of carboxylated modified liposomes and contrast agent
Amount is than being 1:1,1.25:1,1.5:1,1.75:1,2:1,3:1,4:1,5:1,6:1,7:1,8:1,9:1 or 10:1.
By the way that the mass ratio of carboxylated modified liposomes and contrast agent is set as (1-10): 1, to guarantee that carboxylated is modified
Liposome is in excess in contrast agent, avoids the loss of contrast agent；Especially when the matter of carboxylated modified liposomes and contrast agent
Amount is than being (1-2): when 1, carboxylated modified liposomes are higher to the encapsulation rate of contrast agent, can effectively reduce the damage of raw material
It loses.
In the preferred embodiment of the present invention, the mass ratio of carboxylated modified liposomes and lactoferrin is 1:
(1-3), preferably 1:(1.5-2).
In the preferred embodiment of the invention, carboxylated modified liposomes and lactoferrin it is typical but non-limiting
Mass ratio be 1:1,1.25:1,1.5:1,1.75:1,2:1,2.25:1,2.5:1,2.75:1 or 3:1.
Carboxylated modified liposomes are activated by carboxyl activator, so that it is connected with lactoferrin by chemical bond
It connects, in order to improve the reaction efficiency of carboxylated modified liposomes and lactoferrin, the amount of the substance of lactoferrin need to be in excess in carboxylic
The amount of the substance of base activated lipid body, test prove, when the mass ratio of carboxylated modified liposomes and lactoferrin is 1:(1-3)
When, the yield of glioma image nano-probe is higher, the mass ratio especially when carboxylated modified liposomes and lactoferrin
When for 1:(1.5-2), the yield highest of glioma image nano-probe.
It in the preferred embodiment of the present invention, further include carboxylated modified liposomes probe in step (a)
Purifying, purifying include the removal of organic solvent and unentrapped contrast agent.
By by carboxylated modified liposomes probe organic solvent and contrast agent remove, to carboxylated modified lipid
Body probe is purified, and is avoided impurity effect subsequent and is reacted with lactoferrin, and improves the safety of carboxylated modified liposomes
Property and stability.
In present invention further optimization embodiment, organic solvent is volatilized using inert gas purge and is removed.
Inert gas is selected from least one of argon gas, helium and nitrogen, removes organic solvent using inert gas purge
Convenient, safety, it is simple and easy.
In present invention further optimization embodiment, the contrast agent of unentrapped is removed using centrifugation, it is simple and convenient,
It is easily operated.
It in the preferred embodiment of the present invention, further include glioma image nano-probe in step (b)
Purifying, purifying include the removal of carboxyl activator and unreacted lactoferrin.
In present invention further optimization embodiment, using dialysis or ultrafiltration by carboxyl activator and unreacted cream
Ferritin removal.
In the preferred embodiment of the invention, glioma image nano-probe is purified using PBS solution,
Remove activated carboxylic i.e. and unreacted lactoferrin, to improve the safety of glioma image nano-probe provided by the invention
Property, stability and targeting.
In the preferred embodiment of the invention, the purifying of glioma image nano-probe is carried out more using ultrafiltration
Convenient and efficient, used ultrafiltration membrane is 100,000 molecular weight sizes.
According to the third aspect of the present invention, glioma image nano-probe provided by the invention is preparing glioma
Diagnose and/or treat the application in drug.
Technical solution provided by the invention is further described below with reference to embodiment and comparative example.
Embodiment 1
A kind of glioma image nano-probe is present embodiments provided, is prepared in accordance with the following steps:
(1) by 2mg 1,2- distearyl-SN- glycerol -3- phosphatidyl ethanolamine-N- carboxyl-cetomacrogol 1000 (DSPE-
PEG (1000)-COOH) and 1mg ICG be dissolved in 1mL chloroform, after mixing, be added drop-wise in 3mL water, then purge the system with nitrogen,
Until chloroform volatilization is most, it is centrifuged 8000rpm/min, 10min, free ICG is removed, obtains the DSPE-PEG for containing ICG
(2000)-COOH solution；
(2) EDC is added in DSPE-PEG (the 2000)-COOH solution for containing ICG, is activated DSPE-PEG (2000)-
The carboxyl of COOH for 24 hours, with ultrafiltration (100,000 molecular weight sizes) 8000rpm/min, 3min, 6 time, is filled it up with super every time with PBS solution
Chimney filter removes carboxyl activator, finally leaves 2ml sample, then weigh 3.5mg lactoferrin matter, be dissolved in 200ul PBS solution,
It is added in reaction system (2ml), (speed should not be fast) is stirred at room temperature, reaction overnight obtains glioma image nano-probe
Solution.
Embodiment 2
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 1
It is, in step (a), DSPE-PEG (1000)-COOH is substituted using DSPE-PEG (2000)-COOH.
Embodiment 3
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 1
It is, in step (a), DSPE-PEG (1000)-COOH is substituted using DSPE-PEG (3000)-COOH.
Embodiment 4
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 1
It is, in step (a), DSPE-PEG (1000)-COOH is substituted using DSPE-PEG (5000)-COOH.
Embodiment 5
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 1
It is, in step (a), DSPE-PEG (1000)-COOH is substituted using DSPE-PEG (8000)-COOH.
Embodiment 6
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 1
It is, in step (a), DSPE-PEG (1000)-COOH is substituted using DSPE-PEG (10000)-COOH.
Embodiment 7
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 1
It is, in step (a), DSPE-PEG (1000)-COOH is substituted using DSPE-PEG (100)-COOH.
Embodiment 8
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 1
It is, in step (a), DSPE-PEG (1000)-COOH is substituted using DSPE-PEG (200)-COOH.
The glioma image nano-probe solution that embodiment 1-8 is provided is entered into glioma original from tail vein injection respectively
In the nude mice model of position, near-infrared fluorescent/optoacoustic bimodal glioma imaging is carried out, the results show that injection embodiment 1-6 is provided
Glioma image nano-probe nude mice near-infrared fluorescent and photoacoustic imaging than more visible, can compare and accurately identify
The nude mice near-infrared fluorescent for the glioma image nano-probe that the boundary of glioma, especially injection embodiment 2-4 provide
It is apparent with photoacoustic imaging, it is capable of the boundary of more acurrate identification glioma, cuts off clean tumor tissues for operation and help is provided,
And injecting the fluorescence of the nude mice of the glioma image nano-probe of embodiment 7-8 offer and photoacoustic image then cannot clearly see
To glioma boundary, this illustrates in carboxylated modified liposomes, and the degree of polymerization of polyethylene glycol is in 1000-10000, especially
In 2000-5000, glioma image nano-probe can keep good glioma Boundary Recognition function.
It is illustrated below with reference to imaging function of the Fig. 2 and Fig. 3 to glioma image nano-probe, Fig. 2 is to inject this hair
The near-infrared fluorescent figure of the nude mice for the glioma image nano-probe solution that bright embodiment 2 provides；Fig. 3 is that the injection present invention is real
The optoacoustic figure of the nude mice of the glioma image nano-probe of the offer of example 2 is provided；From figures 2 and 3, it will be seen that the injection present invention is real
The near-infrared fluorescent figure and the equal image clearly of optoacoustic figure of the nude mice of the glioma image nano-probe solution of the offer of example 2 are provided,
Clear border, this illustrates that the glioma image nano-probe that the embodiment of the present invention 2 provides can pass through blood-brain barrier, active target
To brain glioblastoma cell, fluorescence and the imaging of optoacoustic bimodal of tumour are realized in the microenvironment of glioma, and can be from figure
It is cut so as to improve the diagnostic accuracy of glioma for human glioma operation on the boundary that glioma is accurately identified as in
Reference is provided except clean.
Embodiment 8
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, in step (2), the quality of lactoferrin is 2mg.
Embodiment 9
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, in step (2), the quality of lactoferrin is 3mg.
Embodiment 10
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, in step (2), the quality of lactoferrin is 4mg.
Embodiment 11
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, in step (2), the quality of lactoferrin is 6mg.
Embodiment 12
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, in step (2), the quality of lactoferrin is 0.5mg.
The glioma image nano-probe solution that embodiment 8-12 is provided is entered into glioma from tail vein injection respectively
In Situ model of nude mice, near-infrared fluorescent/optoacoustic bimodal glioma imaging, and the brain provided with injection embodiment 2 are carried out
The near-infrared fluorescent figure and optoacoustic figure of the nude mice of glioma image nano-probe solution are compared, the results show that injection is implemented
The near-infrared fluorescent and photoacoustic imaging of the nude mice for the glioma image nano-probe that example 8-11 is provided can compare than more visible
Relatively accurately identify the nude mice for the glioma image nano-probe that the boundary of glioma, especially injection embodiment 9-10 are provided
Near-infrared fluorescent and photoacoustic imaging it is apparent, the nude mice of the glioma image nano-probe provided with injection embodiment 2
The clarity of near-infrared fluorescent and photoacoustic imaging is almost the same, is capable of the boundary of more acurrate identification glioma.And it injects and implements
The fluorescence and photoacoustic image of the nude mice for the glioma image nano-probe that example 12 provides are then less clear, and from its fluorescent image
As can be seen that big portion's glioma image nano-probe across blood-brain barrier is less, cause its fluorescence and photoacoustic image at
Image sharpness is poor.This explanation is 1:(1-3 when the mass ratio of carboxylated modified liposomes and lactoferrin) when, made brain glue
The active targeting function of matter tumor image nano-probe is strong, can penetrate blood-brain barrier, monitor the boundary of glioma, cuts for operation
Except clean tumor tissues provide help, especially when the mass ratio of carboxylated modified liposomes and lactoferrin is 1:(1.5-2)
When, made by glioma image nano-probe active targeting it is with better function, blood-brain barrier can be more penetrated, into brain
The boundary of the microenvironment detection tumour of glioma.
Embodiment 13
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, in step (1), the quality of ICG is 0.2mg.
Embodiment 14
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, in step (1), the quality of ICG is 0.5mg.
Embodiment 15
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, in step (1), the quality of ICG is 1.5mg.
Embodiment 16
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, in step (1), the quality of ICG is 2mg.
Embodiment 17
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, in step (1), the quality of ICG is 0.05mg.
Embodiment 18
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, in step (1), the quality of ICG is 10mg.
The glioma image nano-probe solution that embodiment 13-18 is provided is entered into glioma from tail vein injection respectively
In Situ model of nude mice, near-infrared fluorescent/optoacoustic bimodal glioma imaging, and the brain provided with injection embodiment 2 are carried out
The near-infrared fluorescent figure and optoacoustic figure of the nude mice of glioma image nano-probe solution are compared, the results show that injection is implemented
The near-infrared fluorescent and photoacoustic imaging of the nude mice for the glioma image nano-probe that example 13-16 is provided, can than more visible
Compare the glioma image nano-probe that the boundary for accurately identifying glioma, especially injection embodiment 15-16 are provided
The near-infrared fluorescent and photoacoustic imaging of nude mice are apparent, naked with the glioma image nano-probe of the injection offer of embodiment 2
The near-infrared fluorescent of mouse and the clarity of photoacoustic imaging are almost the same, are capable of the boundary of more acurrate identification glioma.And it injects
The fluorescence and photoacoustic image of the nude mice for the glioma image nano-probe that embodiment 17-18 is provided are then less clear, this explanation
When the mass ratio of carboxylated modified liposomes and contrast agent is (1-10): when 1, made glioma image nano-probe
Imaging clearly, especially when the mass ratio of carboxylated modified liposomes and contrast agent be (1-2): when 1, made by brain colloid
The imaging of tumor image nano-probe is apparent.
Embodiment 19
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, used contrast agent is IR-780.
Embodiment 20
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, used contrast agent is IR-797.
Embodiment 21
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, used contrast agent is IR-808.
Embodiment 22
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, used contrast agent is IR-775.
The glioma image nano-probe solution that embodiment 19-22 is provided is entered into glioma from tail vein injection respectively
In Situ model of nude mice, near-infrared fluorescent/optoacoustic bimodal glioma imaging, and the brain provided with injection embodiment 2 are carried out
The near-infrared fluorescent figure and optoacoustic figure of the nude mice of glioma image nano-probe solution are compared, the results show that injection is implemented
The near-infrared fluorescent and photoacoustic imaging of the nude mice for the glioma image nano-probe that example 19-22 is provided, can than more visible
Compare the boundary for accurately identifying glioma, this illustrates that glioma image nano-probe provided by the invention uses different type
Contrast agent can also pass through blood-brain barrier, active targeting brain glioblastoma cell realize tumour in the microenvironment of glioma
Imaging improve the diagnostic accuracy of glioma to accurately identify the boundary of glioma, for human glioma operation cut off
Reference is provided, helps to cut off brain colloid clean.
Embodiment 23
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, in step (1), DSPE-PEG (2000)-COOH is substituted using PC-PEG (2000)-COOH.
Embodiment 24
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, in step (1), DSPE-PEG (2000)-COOH is substituted using DPPC-PEG (2000)-COOH.
Embodiment 25
Present embodiments provide a kind of glioma image nano-probe, the difference of preparation method and embodiment 2
It is, in step (1), DSPE-PEG (2000)-COOH is substituted using PE-PEG (2000)-COOH.
The glioma image nano-probe solution that embodiment 23-25 is provided is entered into glioma from tail vein injection respectively
In Situ model of nude mice, near-infrared fluorescent/optoacoustic bimodal glioma imaging, and the brain provided with injection embodiment 2 are carried out
The near-infrared fluorescent figure and optoacoustic figure of the nude mice of glioma image nano-probe solution are compared, the results show that injection is implemented
The near-infrared fluorescent and photoacoustic imaging of the nude mice for the glioma image nano-probe that example 23-25 is provided, can than more visible
Compare the boundary for accurately identifying glioma, this illustrates that glioma image nano-probe provided by the invention uses different type
The poly ethyldiol modified liposome of carboxylated also can pass through blood-brain barrier, active targeting brain glioblastoma cell, in glioma
Microenvironment in realize tumour imaging improve the diagnostic accuracy of glioma to accurately identify the boundary of glioma,
Reference is provided for human glioma operation excision, helps to cut off brain colloid clean.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
1. a kind of glioma image nano-probe, which is characterized in that including carboxylated modified liposomes, contrast agent and newborn iron egg
White, the hydrophobic layer of the carboxylated modified liposomes contains the contrast agent, the hydrophilic layer of the carboxylated modified liposomes with
For the lactoferrin by chemistry key connection, the contrast agent includes fluorescent contrast agent, photoacoustic contrast agent and fluorescence optoacoustic bimodulus
At least one of state contrast agent, preferably fluorescence optoacoustic bimodal contrast agent.
2. glioma image nano-probe according to claim 1, which is characterized in that the carboxylated modified liposomes
For the poly ethyldiol modified liposome of carboxylated；
Preferably, in the poly ethyldiol modified liposome of carboxylated, the degree of polymerization of polyethylene glycol is 1000-10000, preferably
2000-5000。
3. glioma image nano-probe according to claim 2, which is characterized in that the carboxyl polyethylene glycol changes
Property liposome be selected from the poly ethyldiol modified phosphatidyl choline of carboxylated, the poly ethyldiol modified two palmityls phosphatidyl gallbladder of carboxylated
In the poly ethyldiol modified phosphatidyl-ethanolamine of alkali, carboxylated and the poly ethyldiol modified Distearoyl Phosphatidylcholine of carboxylated extremely
Few one kind.
4. any one of -3 glioma image nano-probe according to claim 1, which is characterized in that the fluorescent contrast agent
Selected from least one of Cy3, Cy5 or Cy5.5；
Preferably, the photoacoustic contrast agent is selected from the molybdenum disulfide or tungsten disulfide of lamella；
Preferably, the fluorescence optoacoustic bimodal contrast agent is selected from indoles cyanine dyes and/or heptamethine cyanine；
Preferably, the indoles cyanine dyes is indocyanine green；
Preferably, the seven methines cyanine dyes is in IR-780, IR-775, IR-797, IR-792, IR-806 or IR-808
At least one.
5. the preparation method of glioma image nano-probe according to claim 1-4, which is characterized in that packet
Include following steps:
(a) carboxylated modified liposomes and contrast agent are added in organic solvent and are uniformly mixed, added in water and be uniformly mixed, make
Contrast agent contains in the hydrophobic layer of carboxylated modified liposomes, obtains liposome probe；
(b) liposome probe is uniformly mixed with carboxyl activator, adds lactoferrin and be uniformly mixed, obtains glioma
Image nano-probe；
Preferably, the organic solvent is volatile organic solvent, preferably in ethyl alcohol, chloroform, methylene chloride and acetone extremely
Few one kind, more preferably ethyl alcohol；
Preferably, carboxyl activator is n-hydroxysuccinimide and/or 1- ethyl -3- (3- dimethyl aminopropyl)-carbonization two
Imines, preferably n-hydroxysuccinimide.
6. the preparation method of glioma image nano-probe according to claim 5, which is characterized in that the carboxylated
The mass ratio of modified liposomes and the contrast agent is (1-10): 1, preferably (1-2): 1.
7. the preparation method of glioma image nano-probe according to claim 5, which is characterized in that the carboxylated
The mass ratio of modified liposomes and the lactoferrin is 1:(1-3), preferably 1:(1.5-2).
8. according to the preparation method of the described in any item glioma image nano-probes of claim 5-7, which is characterized in that
It further include the purifying of liposome probe in step (a), the purifying includes the removal of organic solvent and unentrapped contrast agent；
Preferably, organic solvent is volatilized using inert gas purge and is removed；
Preferably, the contrast agent of unentrapped is removed using centrifugation.
9. according to the preparation method of the described in any item glioma image nano-probes of claim 5-7, which is characterized in that
It further include the purifying of glioma image nano-probe in step (b), the purifying includes carboxyl activator and unreacted cream
The removal of ferritin；
Preferably, carboxyl activator and unreacted lactoferrin are removed using dialysis or ultrafiltration.
10. glioma image nano-probe according to claim 1-4 prepare Diagnosing Gliomas and/or
Treat the application in drug.
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