CN104922671B - Indocyanine green composite nano-particles and preparation method and application thereof - Google Patents
Indocyanine green composite nano-particles and preparation method and application thereof Download PDFInfo
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- CN104922671B CN104922671B CN201510314261.5A CN201510314261A CN104922671B CN 104922671 B CN104922671 B CN 104922671B CN 201510314261 A CN201510314261 A CN 201510314261A CN 104922671 B CN104922671 B CN 104922671B
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Abstract
The invention provides indocyanine green composite nanoparticles, which comprise indocyanine green, hydrophobic polymers, polyethylene glycol-derivatized phospholipids and cancer cell membranes, wherein the indocyanine green is surrounded by the hydrophobic polymers and forms a spheroid structure with the hydrophobic polymers, the polyethylene glycol-derivatized phospholipids are interpenetrated in the cancer cell membranes and form a vesicle structure with the cancer cell membranes, and the spheroid structure is coated in the vesicle structure. The invention also provides a preparation method and application of the indocyanine green composite nano-particles, and when the indocyanine green composite nano-particles are applied, the composite nano-particles have high-efficiency targeting property to tumors, and the indocyanine green is not easy to aggregate and has good stability.
Description
Technical field
The invention belongs to field of nanometer material technology, more particularly to a kind of indocyanine green composite nanometer particle and preparation method thereof and
Using.
Background technology
Indocyanine green (ICG) is the carbonization hydrogen species dyestuff of one kind three, and its maximum absorption wavelength and maximum emission wavelength are in 740nm
With 800nm near infrared region, fluorescence penetration power is strong, is unique a kind of by food and medicine Surveillance Authority of the U.S. (FDA) approval
Be used for physianthropy be imaged and diagnose reagent, while it as a kind of photo-thermal formulation application in clinical thermotherapy.But indoles
Cyanines are green to be assembled rapidly in polar solvent, less stable, and it can quickly be removed in blood plasma, in vivo circulation time
It is short, while indocyanine green also lacks tumour cell targeting specific, these defects greatly limit the application of indocyanine green.
The content of the invention
To solve the above problems, the invention provides a kind of indocyanine green composite nanometer particle and preparation method thereof and answer
With.The stability and targets identification ability of the indocyanine green composite nanometer particle are good, while preparation method technique is simple.
In a first aspect, the invention provides a kind of indocyanine green composite nanometer particle, the indocyanine green composite Nano
Grain includes indocyanine green, hydrophobic polymer, polyglycol derivatization phospholipid and cancer cell membrane, and the indocyanine green is dredged by described
Water-based polymer is surround and forms spherical structure with the hydrophobic polymer, and the polyglycol derivatization phospholipid is interspersed in
Imitated vesicle structure is formed in the cancer cell membrane and with the cancer cell membrane, the near-spherical structure is coated on the imitated vesicle structure
In, the mass ratio of the indocyanine green, hydrophobic polymer and polyglycol derivatization phospholipid is (0.25-1.5)：1：(0.09-
0.27), the cancer cell membrane derives from cancer cell, and the number of the cancer cell and the mass ratio of polyglycol derivatization phospholipid are
(108-109)：180μg.
As described in the present invention, composite shell of the imitated vesicle structure as the indocyanine green composite nanometer particle, use
In the spherical structure for loading the indocyanine green and hydrophobic polymer composition.
Preferably, the hydrophobic polymer be selected from Poly(D,L-lactide-co-glycolide (also known as poly (glycolide-co-lactide)),
The one or more of PLA and polycaprolactone, but not limited to this.
Preferably, the hydrophobic polymer is Poly(D,L-lactide-co-glycolide (being abbreviated as PLGA), the hydrophily
The molecular weight of polymer is 7000-17000, and the copolymerization ratio of monomer lactic acid and hydroxyacetic acid is 50:50.
Preferably, the polyglycol derivatization phospholipid is mutually got continuously by polyethylene glycol by covalent bond with phospholipid substance
Arrive.
Preferably, the molecular weight of the polyethylene glycol is 200~20000.
Preferably, the phospholipid substance can be phosphatide existing for artificial synthesized or nature, the phospholipid thing
Matter can be but be not limited to DSPE, DSPG or cholesterol.
Preferably, the polyglycol derivatization phospholipid is PEG-DSPE (DSPE-
PEG)。
As described in the present invention, the indocyanine green (ICG) and the mass ratio of the hydrophobic polymer are (0.25-
1.5)：1.
Preferably, the indocyanine green and the mass ratio of the hydrophobic polymer are (0.5-1.0)：1.
As described in the present invention, the cancer cell membrane derives from cancer cell.
Preferably, the cancer cell membrane includes one in liver cancer cells, breast cancer cell, lung carcinoma cell, cervical cancer cell
Kind is a variety of, but not limited to this.
As described in the present invention, the mass ratio of the hydrophobic polymer and the polyglycol derivatization phospholipid is 1：
(0.09-0.27)。
Preferably, the mass ratio of the hydrophobic polymer and the polyglycol derivatization phospholipid is 1：(0.15-
0.21)。
Preferably, the particle diameter of the indocyanine green composite nanometer particle is 90-180nm.
As described in the present invention, the particle diameter is measured using transmission electron microscope.
The indocyanine green composite nanometer particle (being abbreviated as CCINPs) that first aspect present invention provides, the indoles cyanines
Green composite nanometer particle includes indocyanine green, hydrophobic polymer, polyglycol derivatization phospholipid and cancer cell membrane, the poly- second
Phosphatide in glycol derivatization phospholipid is combined so as to be interspersed in by physical action with the phospholipid molecule in the cancer cell membrane
State in cancer cell membrane, certain support, fixation played to cancer cell membrane, and imitated vesicle structure is formed with the cancer cell membrane,
The imitated vesicle structure has hydrophilic composite shell and hydrophobic cavity；The indocyanine green is by the hydrophobic polymer
It surround and forms spherical structure with the hydrophobic polymer, hydrophobic polymer wraps up hydrophilic indocyanine green, described
The hydrophobic cavity that hydrophily composite shell is formed is used to load ICG and hydrophobic polymer, it is possible to prevente effectively from ICG occurs
Aggregation.
The indocyanine green composite nanometer particle provided by the invention, on the one hand, the cancer cell membrane is exposed to described multiple
The outermost layer of shell is closed, due to containing the molecule with cancer cell energy targets identification on the cancer cell membrane, such as folacin receptor, first tire
A variety of biological recognition ligands such as albumen, E-Cadherin, galectin-3, and homophilic adhesion can be based on corresponding tumour cell
Act on and be mutually distinguishable, the synergy based on multiple ligands, the indocyanine green composite nanometer particle can be with tumour cell
Specific binding is formed, there is higher targeting to tumour cell, indocyanine green is carried well and enters into the cell, can improve
Indocyanine green improves the oncotherapy effect of indocyanine green to tumour cell targeting specific.On the other hand, it is described hydrophilic compound
PEG in shell can effectively hinder identification of the immune system to the nano particle, significantly extend the nano particle in body
Interior circulation time, and then be enriched to by enhancing infiltration retention effect (EPR effects) in tumor tissues, finally realize tumour
Passive target.Passive target caused by active targeting and PEG based on above-mentioned cancer cell membrane, the indocyanine green composite Nano
Grain has very strong affinity to tumour cell.
Homologous absorption of the present invention, refer to refer to intercellular adhesion, be a kind of form of cell-tocell exchange, and
The solvable mediator of information interchange claims cell adhesion molecule (CAM).Cell adhesion molecule is that one kind has independent molecular structure
Material, mutual adhesion can occur by identifying the specific receptor adhered to it.Two adjacent cell surfaces
Being mutually distinguishable between allogenic cell adhesion molecule and combine, as homologous absorption.
Therefore, indocyanine green composite nanometer particle provided by the invention can improve ICG stability, can effectively avoid
ICG decomposition and in vivo removing, while the targets identification effect to tumour can be dramatically increased, tumour can be advantageously applied to
Thermotherapy.
The indocyanine green composite nanometer particle has good biocompatibility, can be by biodegradation and by normal
Physiological pathway absorb or excrete, to organism injure it is small.
The indocyanine green, hydrophobic polymer, the mass ratio of polyglycol derivatization phospholipid and cancer cell membrane are
(0.25-1.5)：1：(0.09-0.27), the number of the cancer cell and the mass ratio of polyglycol derivatization phospholipid are 108-
109：180 μ g, under the mass ratio, nanoparticle structure can be formed between each component well, states indocyanine green composite Nano
The particle diameter distribution of particle is more uniform, and average grain diameter 90-180nm, pattern is relatively regular, favorable dispersibility avoids indocyanine green
Aggregation, improve the stability and targeting of indocyanine green.
Second aspect, the invention provides a kind of preparation method of indocyanine green composite nanometer particle, comprise the following steps：
(1) cancer cell is taken, extracts cancer cell membrane；
(2) imitated vesicle structure is prepared：Cancer cell membrane after extraction is pressed to the number and polyglycol derivatization phospholipid of cancer cell
Than for (108-109)：180 μ g are weighed, and are dissolved in the first solvent, the ultrasonic 3-6min under ice bath, it is molten to obtain the first mixing
Liquid；
(3) spherical kernel is prepared：Indocyanine green is dissolved in the second solvent and obtains indocyanine green solution, hydrophobicity is more
Polymers is dissolved in the 3rd solvent and obtains hydrophobic polymer solution, and hydrophobic polymer solution is added dropwise into indocyanine green solution
In, ultrasonic 2-4min is carried out with ultrasonic wave, obtains the second mixed solution；
(4) cancer cell vesicle solution described in step (2) is added in step (3) second mixed solution, ultrasound
2-4min is mixed, obtains the 3rd mixed solution, the 3rd mixed solution is subjected to centrifugal treating, supernatant is collected and obtains indoles
The green composite nanometer particle of cyanines, the indocyanine green composite nanometer particle include indocyanine green, hydrophobic polymer, polyethylene glycol and spread out
Biochemical phosphatide and cancer cell membrane, the indocyanine green are surround and formed with the hydrophobic polymer by the hydrophobic polymers
Spherical structure, the polyglycol derivatization phospholipid are interspersed in the cancer cell membrane and form vesica with the cancer cell membrane
Structure, the near-spherical structure are coated in the imitated vesicle structure, wherein, the indocyanine green, hydrophobic polymer and poly- second
The mass ratio of glycol derivatization phospholipid is (0.25-1.5)：1:(0.09-0.27).
Preferably, in step (1), the extraction cancer cell membrane is specially：Postdigestive cancer cell is taken, adds pH=7.5
Tris-HCl hypotonic buffer solution, homogeneous is broken, low-speed centrifugal 5-10min, collects supernatant, high speed centrifugation 15-30min,
Supernatant is regathered, then ultracentrifugation 40-60min is carried out to supernatant, collects precipitation, and precipitation is cleaned with cushioning liquid,
Obtain cancer cell membrane after purification.
Preferably, in step (1), the centrifugal force of the low-speed centrifugal is 3200g, and the ultracentrifugal centrifugal force is
20000g, the centrifugal force of the ultracentrifugation is 100000g.
As described in the present invention, for different cancer cells, different pH, various concentrations, the hypotonic buffer of species may be selected
Solution.
The hypotonic buffer solution is advantageous to the separation of cell membrane and cellular content, is easy to extract cancer cell membrane.
Preferably, when the cancer cell is human breast cancer cell, the hypotonic buffer solution also includes KCl, MgCl2、
Protease inhibitors, KCl concentration is 10mM, MgCl2Concentration be 2mM, the concentration of protease inhibitors be 0.06~2.0 μ
g/ml。
As described in the present invention, during composite nanometer particle is prepared, broken using the ultrasound of low temperature ice bath, low-power
It is broken, the damage to albumen on cancer cell membrane can be reduced as far as possible, can retain certain ligand activity.
Preferably, in step (2), first solvent is the one or more of ethanol, water and methanol.
It is further preferred that in step (2), first solvent is the ethanol water that mass fraction is 4%.
Preferably, in step (3), second solvent is the one or more in ethanol, water and methanol.
It is further preferred that in step (2), first solvent is the ethanol water that mass fraction is 4%.
Preferably, in step (3), the 3rd solvent is the one or more in acetonitrile and acetone.
As described in the present invention, the 3rd solvent is the amphiphilic solvent that can dissolve hydrophobic polymer.
As described in the present invention, the indocyanine green and the mass ratio of the hydrophobic polymer are (0.25-1.5)：1.
Preferably, the indocyanine green and the mass ratio of the hydrophobic polymer are (0.5-1.0)：1.
As described in the present invention, the mass ratio of the hydrophobic polymer and the polyglycol derivatization phospholipid is 1：
(0.09-0.27)。
Preferably, the mass ratio of the hydrophobic polymer and the polyglycol derivatization phospholipid is 1：(0.15-
0.21)。
Preferably, in step (3), the mass concentration of the indocyanine green solution is 0.5mg/ml.
Preferably, in step (3), the concentration of the hydrophobic polymer solution is 1mg/ml.
Preferably, the volume ratio of the indocyanine green solution and the hydrophobic polymer solution is 1.5:1.
As described in the present invention, in step (4), hydrophobic polymer solution is added dropwise in indocyanine green solution,
To ensure that ultrasound can be carried out fully, finally the dispersiveness of the indocyanine green composite nanometer particle of gained is preferable, particle diameter is smaller.
Preferably, in step (4), centrifugal treating ultrafiltration 2-3 in the ultra-filtration centrifuge tube that molecular cut off is 10kDa
It is secondary.
Preferably, in step (4), the centrifugal treating is under centrifugal rotational speed 6000-7000rpm, centrifuges 1-3min.
As described in the present invention, centrifugal treating described in step (4), short time can avoid cancer cell membrane adherent, repeatedly may be used
Solvent is fully removed, centrifugal speed is too high or too low, all the form of final nano particle can be made a big impact, the application is
1-3min is centrifuged under 6000-7000rpm, and repeats centrifugal treating 2-3 times.
Preferably, ultrasonic cell disruption instrument is used with 20kHz frequency and 35W power in step (2), (3), (4)
Carry out ultrasound.
Preferably, the particle diameter of the indocyanine green composite nanometer particle is 90-180nm.
Preferably, the polyglycol derivatization phospholipid is mutually got continuously by polyethylene glycol by covalent bond with phospholipid substance
Arrive.
Preferably, the molecular weight of the polyethylene glycol is 200~20000.
Preferably, the phospholipid substance can be phosphatide existing for artificial synthesized or nature, the phospholipid thing
Matter can be but be not limited to DSPE, DSPG or cholesterol.
Preferably, the polyglycol derivatization phospholipid is PEG-DSPE (DSPE-
PEG)。
Preferably, the cancer cell membrane includes one in liver cancer cells, breast cancer cell, lung carcinoma cell, cervical cancer cell
Kind is a variety of, but not limited to this.The cancer cell membrane is from the animal cancer cell without cell membrane.
According to the cancer cell membrane and the chemical property of polyglycol derivatization phospholipid after purification, hydrophily is dissolved them in
Solvent, according to the similar principle to mix, the phosphatide in the polyglycol derivatization phospholipid is thin by physical action and the cancer
Phospholipid molecule in after birth certain supported, fixed made with reference to so as to be interspersed in the cancer cell membrane, being played to cancer cell membrane
With and being assembled into imitated vesicle structure with the cancer cell membrane, the imitated vesicle structure has hydrophilic composite shell and hydrophobic
Cavity.
The hydrophobic polymer is green around the small molecule indole cyanines by self assembly effect, beneficial to the scattered of ICG, institute
State the hydrophobicity cavity in imitated vesicle structure to can be used for loading hydrophobic polymer and small molecule ICG, it is possible to prevente effectively from ICG occurs
Aggregation.The cancer cell membrane provides polyethylene glycol hydrophily outer layer and energy and tumour for the indocyanine green composite nanometer particle
With reference to associated ligands.
The indocyanine green, hydrophobic polymer and cancer cell membrane and polyglycol derivatization phospholipid pass through self assembling process
Form the indocyanine green composite nanometer particle, it is not necessary to chemically reacted, preparation process is environment-protecting and non-poisonous, and preparation method is simple
It is easy to operate.Resulting indocyanine green composite nanometer particle has the function that optical diagnostics and treating cancer simultaneously.
The third aspect, the invention provides a kind of indocyanine green composite nanometer particle answering in tumor thermotherapy medicine is prepared
With.
The indocyanine green composite nanometer particle is made using the preparation method described in second aspect of the present invention.
Preferably, the particle diameter of the indocyanine green composite nanometer particle is 90-180nm.
Indocyanine green composite nanometer particle provided by the invention can improve the targets identification to tumour, while can improve
ICG stability, it can effectively avoid ICG decomposition and remove in vivo, the thermotherapy of tumour can be applied well.
To sum up, beneficial effects of the present invention include the following aspects：
(1) indocyanine green composite nanometer particle produced by the present invention can effectively improve targeting of the indocyanine green to tumour
Property, while the aggregation of indocyanine green can be prevented, the stability of indocyanine green is improved, extends its circulation time in vivo；
(2) preparation method of heretofore described indocyanine green composite nanometer particle is simple and easy, easy to operation；
(3) the indocyanine green composite nanometer particle produced by the present invention can be used for preparing tumor thermotherapy medicine.
Brief description of the drawings
Fig. 1 is the structural representation of indocyanine green composite nanometer particle prepared by embodiment 1；
Fig. 2 is the high-resolution-ration transmission electric-lens figure of indocyanine green composite nanometer particle prepared by embodiment 1；
Fig. 3 is the grain size distribution of indocyanine green composite nanometer particle prepared by embodiment 1；
The cell endocytic result figure for the indocyanine green composite nanometer particle (CCINPs) that Fig. 4 is prepared for embodiment 1, first row
Experimental group is classified as control group, second；
Fig. 5 is the design sketch that indocyanine green composite nanometer particle is used for the experiment of animal targeting in Application Example.
Embodiment
As described below is the preferred embodiment of the present invention, it is noted that for those skilled in the art
For, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications are also considered as
Protection scope of the present invention.
Embodiment 1
A kind of preparation method of indocyanine green composite nanometer particle, comprises the following steps：
(1) cancer cell membrane is extracted：Cancer cell is taken, adds hypotonic buffer solution, homogeneous crushes, and precipitation is repeatedly collected by centrifugation,
That is cancer cell membrane；
Human breast cancer cell (MCF-7) is inoculated in T-175 Tissue Culture Flasks, cultivated with DMEM culture mediums, and is added
Enter 10%FBS, 1% penicillin, 1% streptomysin, after cell covers with, discard outmoded culture medium, digested with EDTA, is used in combination
PBS centrifugations, washing 3 times, obtain targeted cancerous cells；
20mM, pH=7.5 Tris-HCl hypotonic buffer liquid, the hypotonic buffer are added toward the MCF-7 cells after washing
Solution also KCl, 2mM comprising 10mM MgCl2, 1.0 μ g/ml protease inhibitors, it is equal with Dounce homogenizer
Matter machine is broken 20 times, the low-speed centrifugal 5min under 3200g centrifugal force to cell homogeneous, preserves supernatant, and add toward precipitation
Hypotonic buffer liquid, then homogeneous is broken, low-speed centrifugal, collects supernatant twice；By above-mentioned supernatant under 20000g centrifugal force
High speed centrifugation 20min, precipitation is abandoned, then supernatant is subjected to ultracentrifugation 50min under 100000g centrifugal force, finally
Obtained pelletizing membrane material precipitation is cleaned one time with 10mM, pH=7.5 Tris-HCL buffer solutions (EDTA containing 1mM), is obtained
Cancer cell membrane after purification；
(2) imitated vesicle structure is prepared：Number and distearyl by the cancer cell membrane after extraction by the cancer cell in its source
The mass ratio of phosphatidyl-ethanolamine-polyethylene glycol (DSPE-PEG) (wherein PEG molecular weight is 2000) is 6 × 108：180μg
Weighed, and be dissolved in 0.5ml, 4% ethanol, use ultrasonic cell disruption instrument with 20kHz frequency under ice bath and
35W power carries out ultrasonic 5min, obtains the first mixed solution；
(3) spherical kernel is prepared：Indocyanine green (ICG) is dissolved in ethanol and obtains 0.5mg/ml ICG solution, will be poly-
Poly lactic coglycolic acid (PLGA) is dissolved in acetonitrile and obtains 1mg/ml PLGA solution, takes 1.5ml ICG solution, and to it
Ultrasound is carried out, while 1ml PLGA solution is added dropwise in 1.5ml ICG solution, ultrasonic time 2min, obtains second
Mixed solution；
(4) the first mixed solution described in 0.5ml steps (2) is added to 2.5ml steps (3) second mixed solution
In, ultrasonic 2min is carried out with 20kHz frequency and 35W power with ultrasonic cell disruption instrument, obtains the 3rd mixed solution, will
3rd mixed solution carries out centrifugal treating, and the centrifugation is the ultrafiltration in the ultra-filtration centrifuge tube that molecular cut off is 10kDa
2-3 times, centrifugal rotational speed is centrifugation time 2min under 6000rpm, collects supernatant and obtains indocyanine green composite nanometer particle
(CCINPs), the indocyanine green composite nanometer particle includes indocyanine green (ICG), hydrophobic polymer PLGA, polyethylene glycol
Derivatization phospholipid DSPE-PEG and cancer cell membrane, the indocyanine green ICG by the hydrophobic polymer PLGA surround and with institute
State hydrophobic polymer PLGA and form spherical structure, it is thin that the polyglycol derivatization phospholipid DSPE-PEG is interspersed in the cancer
Imitated vesicle structure is formed in after birth and with the cancer cell membrane, the near-spherical structure is coated in the imitated vesicle structure.
Fig. 1 is the structural representation of indocyanine green composite nanometer particle (CCINPs) prepared by embodiment 1；11 be Yin in figure
Diindyl cyanines are green, 12 be hydrophobic polymer PLGA, and 1 is the 11 and 12 spherical structures formed；21 be polyglycol derivatization phospholipid
DSPE-PEG, 22 be cancer cell membrane, and 2 be 21 and 22 imitated vesicle structures collectively formed, wherein there be polysaccharide, cell adherence in 22 surfaces
Molecule etc. (is not drawn into) here, and in Fig. 1,11 are represented with the rhombus inside CCINPs, and 12 are represented with the curve inside CCINPs, and 11
The ICG of expression is surround by 12 and is formed spherical structures 1,21 with 12 and represented with the curve outside CCINPs, 21 be interspersed in it is described
Imitated vesicle structure is formed in cancer cell membrane 22 and with the cancer cell membrane 22, the near-spherical structure is coated on the imitated vesicle structure
In.
Fig. 2 is the transmission electron microscope picture of indocyanine green composite nanometer particle (CCINPs) made from embodiment 1, can from Fig. 2
To find out, the particle diameter of indocyanine green composite nanometer particle (CCINPs) made from the present embodiment is 150nm or so, outer layer in Fig. 2
The deeper imitated vesicle structure formed for cancer cell membrane and DSPE-PEG of color, inner side colourity is shallower to be formed for ICG and PLGA
Spherical structure.
Fig. 3 is the grain that indocyanine green composite nanometer particle (CCINPs) made from embodiment 1 is measured using dynamic light scattering
Footpath distribution map, from figure 3, it can be seen that the particle diameter distribution of CCINPs made from the present embodiment is more uniform.
Embodiment 2
A kind of preparation method of indocyanine green composite nanometer particle, comprises the following steps：
(1) cancer cell membrane is extracted：Step is the same as embodiment 1；
(2) imitated vesicle structure is prepared：Number and DSPE-PEG by the cancer cell membrane after extraction by the cancer cell in its source
The ratio of (wherein PEG molecular weight is 400) is 3 × 108：180 μ g are weighed, and are dissolved in 1ml, 4% ethanol, in ice bath
It is lower to use ultrasonic cell disruption instrument to carry out ultrasonic 5min with 20kHz frequency and 35W power, obtain the first mixed solution；
(3) spherical kernel is prepared：Indocyanine green (ICG) is dissolved in ethanol and obtains 0.5mg/ml ICG solution, will be poly-
Poly lactic coglycolic acid (PLGA) is dissolved in acetonitrile and obtains 0.5mg/ml PLGA solution, takes 1.5ml ICG solution, and right
It carries out ultrasound, while 1ml PLGA solution is added dropwise in 1.5ml ICG solution, ultrasonic time 2min, obtains the
Two mixed solutions；
(4) the first mixed solution described in 0.75ml steps (2) is added in step (3) second mixed solution,
Ultrasonic 2min is carried out with 20kHz frequency and 35W power with ultrasonic cell disruption instrument, the 3rd mixed solution is obtained, by institute
State the 3rd mixed solution and carry out centrifugal treating, the centrifugation is the ultrafiltration 2-3 in the ultra-filtration centrifuge tube that molecular cut off is 10kDa
Secondary, centrifugal rotational speed is centrifugation time 2min under 6500rpm, collects supernatant and obtains indocyanine green composite nanometer particle
(CCINPs), the indocyanine green composite nanometer particle includes indocyanine green (ICG), hydrophobic polymer PLGA, polyethylene glycol
Derivatization phospholipid DSPE-PEG and cancer cell membrane, the indocyanine green ICG by the hydrophobic polymer PLGA surround and with institute
State hydrophobic polymer PLGA and form spherical structure, it is thin that the polyglycol derivatization phospholipid DSPE-PEG is interspersed in the cancer
Imitated vesicle structure is formed in after birth and with the cancer cell membrane, the near-spherical structure is coated in the imitated vesicle structure.
Embodiment 3
A kind of preparation method of indocyanine green composite nanometer particle, comprises the following steps：
(1) cancer cell membrane is extracted：Step is the same as embodiment 1；
(2) imitated vesicle structure is prepared：Number and DSPE-PEG by the cancer cell membrane after extraction by the cancer cell in its source
The ratio of (wherein PEG molecular weight is 20000) is 9 × 108：180 μ g are weighed, and are dissolved in 1ml, 4% ethanol, in ice
Bath is lower to use ultrasonic cell disruption instrument to carry out ultrasonic 5min with 20kHz frequency and 35W power, and it is molten to obtain the first mixing
Liquid；
(3) spherical kernel is prepared：Indocyanine green (ICG) is dissolved in ethanol and obtains 0.5mg/ml ICG solution, will be poly-
Poly lactic coglycolic acid (PLGA) is dissolved in acetonitrile and obtains 2mg/ml PLGA solution, takes 1.5ml ICG solution, and to it
Ultrasound is carried out, while 1ml PLGA solution is added dropwise in 1.5ml ICG solution, ultrasonic time 2min, obtains second
Mixed solution；
(4) the first mixed solution described in step (2) is added in step (3) second mixed solution, with ultrasound
Ripple cell crushing instrument carries out ultrasonic 2min with 20kHz frequency and 35W power, the 3rd mixed solution is obtained, by the described 3rd
Mixed solution carries out centrifugal treating, and the centrifugation is ultrafiltration 2-3 time in the ultra-filtration centrifuge tube that molecular cut off is 10kDa, from
Heart rotating speed is centrifugation time 1min under 7000rpm, collects supernatant and obtains indocyanine green composite nanometer particle (CCINPs),
The indocyanine green composite nanometer particle includes indocyanine green (ICG), hydrophobic polymer PLGA, polyglycol derivatization phospholipid
DSPE-PEG and cancer cell membrane, the indocyanine green ICG are circular and more with the hydrophobicity by the hydrophobic polymer PLGA
Polymers PLGA forms spherical structure, the polyglycol derivatization phospholipid DSPE-PEG be interspersed in the cancer cell membrane and with
The cancer cell membrane forms imitated vesicle structure, and the near-spherical structure is coated in the imitated vesicle structure.
Embodiment 4
A kind of preparation method of indocyanine green composite nanometer particle, comprises the following steps：
(1) cancer cell membrane is extracted：Step is the same as embodiment 1；
(2) imitated vesicle structure is prepared：By number of the cancer cell membrane after extraction by the cancer cell in its source and poly- DSPE-
PEG (wherein PEG molecular weight is 7000) mass ratio is 6 × 108：180 μ g are weighed, and are dissolved in 1ml, 4% second
Alcohol, use ultrasonic cell disruption instrument to carry out ultrasonic 5min with 20kHz frequency and 35W power under ice bath, obtain first
Mixed solution；
(3) spherical kernel is prepared：Indocyanine green (ICG) is dissolved in ethanol and obtains 0.5mg/ml ICG solution, will be poly-
Poly lactic coglycolic acid (PLGA) is dissolved in acetonitrile and obtains 1mg/ml PLGA solution, takes 1.5ml ICG solution, and to it
Ultrasound is carried out, while 1ml PLGA solution is added dropwise in 1.5ml ICG solution, ultrasonic time 2min, obtains second
Mixed solution；
(4) the first mixed solution described in 833 μ l steps (2) is added to 2.5ml steps (3) second mixed solution
In, ultrasonic 2min is carried out with 20kHz frequency and 35W power with ultrasonic cell disruption instrument, obtains the 3rd mixed solution, will
3rd mixed solution carries out centrifugal treating, and the centrifugation is the ultrafiltration in the ultra-filtration centrifuge tube that molecular cut off is 10kDa
2-3 times, centrifugal rotational speed is centrifugation time 2min under 6000rpm, collects supernatant and obtains indocyanine green composite nanometer particle
(CCINPs), the indocyanine green composite nanometer particle includes indocyanine green (ICG), hydrophobic polymer PLGA, polyethylene glycol
Derivatization phospholipid DSPE-PEG and cancer cell membrane, the indocyanine green ICG by the hydrophobic polymer PLGA surround and with institute
State hydrophobic polymer PLGA and form spherical structure, it is thin that the polyglycol derivatization phospholipid DSPE-PEG is interspersed in the cancer
Imitated vesicle structure is formed in after birth and with the cancer cell membrane, the near-spherical structure is coated in the imitated vesicle structure.
Embodiment 5
A kind of preparation method of indocyanine green composite nanometer particle, comprises the following steps：
(1) cancer cell membrane is extracted：Step is the same as embodiment 1；
(2) imitated vesicle structure is prepared：By number of the cancer cell membrane after extraction by the cancer cell in its source and poly- DSPE-
PEG (wherein PEG molecular weight is 7000) mass ratio is 6 × 108：180 μ g are weighed, and are dissolved in 1ml, mass fraction
For 4% ethanol, ultrasonic cell disruption instrument is used to carry out ultrasonic 5min with 20kHz frequency and 35W power under ice bath,
Obtain the first mixed solution；
(3) spherical kernel is prepared：Indocyanine green (ICG) is dissolved in ethanol and obtains 0.5mg/ml ICG solution, will be poly-
Poly lactic coglycolic acid (PLGA) is dissolved in acetonitrile and obtains 0.5mg/ml PLGA solution, takes 1.5ml ICG solution, and right
It carries out ultrasound, while 1ml PLGA solution is added dropwise in 1.5ml ICG solution, ultrasonic time 2min, obtains the
Two mixed solutions；
(4) the first mixed solution described in 583 μ l steps (2) is added to 2.5ml steps (3) second mixed solution
In, ultrasonic 2min is carried out with 20kHz frequency and 35W power with ultrasonic cell disruption instrument, obtains the 3rd mixed solution, will
3rd mixed solution carries out centrifugal treating, and the centrifugation is the ultrafiltration in the ultra-filtration centrifuge tube that molecular cut off is 10kDa
2-3 times, centrifugal rotational speed is centrifugation time 2min under 6000rpm, collects supernatant and obtains indocyanine green composite nanometer particle
(CCINPs), the indocyanine green composite nanometer particle includes indocyanine green (ICG), hydrophobic polymer PLGA, polyethylene glycol
Derivatization phospholipid DSPE-PEG and cancer cell membrane, the indocyanine green ICG by the hydrophobic polymer PLGA surround and with institute
State hydrophobic polymer PLGA and form spherical structure, it is thin that the polyglycol derivatization phospholipid DSPE-PEG is interspersed in the cancer
Imitated vesicle structure is formed in after birth and with the cancer cell membrane, the near-spherical structure is coated in the imitated vesicle structure.
The cell endocytic experiment of the indocyanine green composite nanometer particle (CCINPs) of Application Example 1
Cell endocytic experiment is carried out to indocyanine green composite nanometer particle (CCINPs) made from the embodiment of the present invention 1, with
Free ICG is as follows as control, operating procedure：
(1) MCF-7 breast cancer cells are inoculated in 2 group of 8 porocyte culture plates, per hole 2 × 104Individual small cell, per hole
The μ L of culture medium 200 DMEM culture mediums；After cell culture 24h is adherent, the DMEM culture mediums that more renew, into every hole of experimental group
The CCINPs that 200 μ l contain 18 μ g/ml ICG is added, the free ICG that 200 μ l contain 18 μ g/mL is added into every hole of control group, after
Continuous culture 2 hours；
(2) after cultivating 2 hours, the supernatant in orifice plate is sopped up, cell is rinsed with PBS (phosphate buffer), is used in combination
The dyestuffs of Hochest 33258 contaminate nucleus, finally 8 orifice plates are placed in into laser confocal microscope
Observed under (Leica TCS SP5 models) and shoot photo.
The experimental result of tumor-targeting is as shown in figure 4, blueness represents nucleus Hoeches 33258 fluorescence, red
ICG fluorescence is represented, the first of Fig. 4 is classified as free ICG cell endocytic result, and the second of Fig. 4 is classified as the intracellular of CCINPs
Result is gulped down, due to the cladding of the ICG composite shells being made up of cancer cell membrane, shown CCINPs nano particles can effectively improve
Indocyanine green can prevent ICG aggregation to the targeting of tumour cell, improve ICG stability, extend it in vivo
Circulation time, thus in Fig. 2 secondary series ICG red fluorescent apparently higher than the control group of first row, result above table
Bright, CCINPs has efficient tumor-targeting compared to free ICG.
Application Example 2CCINPs animal targeting experiment
1 × 10 is subcutaneously injected in two groups of female BAl BIcs/c nude mices within (4-6 weeks, body weight is 15-20mg)6Individual MCF-7 breast cancer
Cell, treat that gross tumor volume reaches 50mm3(gross tumor volume calculation formula is during left and right：Length of tumor × (tumor width)2/ 2), exist
One of which nude mice tail vein injection 0.2ml CCINPs (ICG concentration is 0.05mg/kg), it is naked as experimental group, another set
Tail vein injection 0.2ml dissociates indocyanine green (ICG) (ICG injected in two groups quality is the same) as a control group,
0.5th, taken pictures after 6,24 hours under small animal living body imager observation, as a result as shown in Figure 5.
In Fig. 5, in figure circled represent knub position, dissociate ICG injection 30 minutes after, be predominantly located at liver, 6h,
24h fluorescence weakens rapidly, and its reason is free ICG less stables, and it can quickly be removed in blood plasma, when circulating in vivo
Between it is short, while indocyanine green lacks tumour cell targeting specific, and knub position do not have ICG enrichments；
Indocyanine green composite nanometer particle (CCINPs) is located at the liver and tumor vicinity of mouse, tumour after injecting 30 minutes
Region its fluorescence signal after CCINPs6h and 24h is injected gradually strengthens, and further confirms that CCINPs can be notable after wrapping up ICG
Reduce the medicines of ICG in vivo to remove and degrade, while close same sex adhesive attraction and tumour EPR by being coated with cancer cell membrane are imitated
Passive target effect caused by answering, significantly improve the Targeting Effect of medicine.
It these results suggest that, when indocyanine green composite nanometer particle (CCINPs) provided by the invention is applied to animal carcasses
Targeting it is preferable.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (9)
1. a kind of indocyanine green composite nanometer particle, it is characterised in that the indocyanine green composite nanometer particle includes indoles cyanines
Green, hydrophobic polymer, polyglycol derivatization phospholipid and cancer cell membrane, the indocyanine green is by the hydrophobic polymer ring
Spherical structure is formed around and with the hydrophobic polymer, the polyglycol derivatization phospholipid is interspersed in the cancer cell membrane
In and with the cancer cell membrane form imitated vesicle structure, the near-spherical structure is coated in the imitated vesicle structure, the indoles cyanines
The mass ratio of green, hydrophobic polymer and polyglycol derivatization phospholipid is (0.25-1.5)：1：(0.09-0.27), the cancer
Cell membrane derives from cancer cell, and the number of the cancer cell and the mass ratio of polyglycol derivatization phospholipid are (108-109)：180
μg；The particle diameter of the indocyanine green composite nanometer particle is 90-180nm.
2. indocyanine green composite nanometer particle as claimed in claim 1, it is characterised in that the hydrophobic polymer with it is described
The mass ratio of polyglycol derivatization phospholipid is 1：(0.15-0.21).
3. indocyanine green composite nanometer particle as claimed in claim 1, it is characterised in that it is thin that the cancer cell membrane includes liver cancer
One or more in born of the same parents, breast cancer cell, lung carcinoma cell, cervical cancer cell, the hydrophobic polymer are selected from polylactic acid-glycolic
The one or more of acetic acid copolymer, PLA and polycaprolactone, the polyglycol derivatization phospholipid lead to for polyethylene glycol
Cross covalent bond to be connected to obtain with phospholipid substance, the molecular weight of the polyethylene glycol is 200~20000.
4. a kind of preparation method of indocyanine green composite nanometer particle, it is characterised in that comprise the following steps：
(1) cancer cell is taken, extracts cancer cell membrane；
(2) imitated vesicle structure is prepared：It is with polyglycol derivatization phospholipid ratio by the number of cancer cell by the cancer cell membrane after extraction
(108-109)：180 μ g are weighed, and are dissolved in the first solvent, the ultrasonic 3-6min under ice bath, obtain the first mixed solution；
(3) spherical kernel is prepared：Indocyanine green is dissolved in the second solvent and obtains indocyanine green solution, by hydrophobic polymer
It is dissolved in the 3rd solvent and obtains hydrophobic polymer solution, hydrophobic polymer solution is added dropwise in indocyanine green solution,
Ultrasonic 2-4min is carried out with ultrasonic wave, obtains the second mixed solution；
(4) cancer cell vesicle solution described in step (2) is added in step (3) second mixed solution, ultrasonic mixing
2-4min, the 3rd mixed solution is obtained, the 3rd mixed solution is subjected to centrifugal treating, collected supernatant and obtain indocyanine green
Composite nanometer particle, the indocyanine green composite nanometer particle include indocyanine green, hydrophobic polymer, polyglycol derivatization
Phosphatide and cancer cell membrane, the indocyanine green are surround by the hydrophobic polymer and form class ball with the hydrophobic polymer
Shape structure, the polyglycol derivatization phospholipid are interspersed in the cancer cell membrane and form vesica knot with the cancer cell membrane
Structure, the near-spherical structure are coated in the imitated vesicle structure, wherein, the indocyanine green, hydrophobic polymer and poly- second two
The mass ratio of alcohol derivatization phospholipid is (0.25-1.5)：1：(0.09-0.27)；The particle diameter of the indocyanine green composite nanometer particle
For 90-180nm.
5. the preparation method of indocyanine green composite nanometer particle as claimed in claim 4, it is characterised in that in step (1), institute
Stating extraction cancer cell membrane is specially：Postdigestive cancer cell is taken, adds pH=7.5 Tris-HCl hypotonic buffer solution, homogeneous
Broken, low-speed centrifugal 5-10min, supernatant is collected, high speed centrifugation 15-30min, regathers supernatant, then supernatant is carried out
Ultracentrifugation 40-60min, precipitation is collected, and precipitation is cleaned with cushioning liquid, obtain cancer cell membrane after purification, wherein institute
The centrifugal force for stating low-speed centrifugal is 3200g, and the ultracentrifugal centrifugal force is 20000g, the centrifugal force of the ultracentrifugation
For 100000g.
6. the preparation method of indocyanine green composite nanometer particle as claimed in claim 4, it is characterised in that first solvent
For the one or more in ethanol, first alcohol and water, second solvent is the one or more in ethanol, water or methanol, described
3rd solvent is the one or more in acetonitrile and acetone.
7. the preparation method of indocyanine green composite nanometer particle as claimed in claim 4, it is characterised in that the hydrophobicity is more
The mass ratio of polymers and the polyglycol derivatization phospholipid is 1：(0.15-0.21).
8. the preparation method of indocyanine green composite nanometer particle as claimed in claim 4, it is characterised in that step (2),
(3), ultrasound described in (4) is to use ultrasonic cell disruption instrument to be carried out with 20kHz frequency and 35W power.
9. a kind of indocyanine green composite nanometer particle as described in claim any one of 1-3 is in tumor thermotherapy medicine is prepared
Using.
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