CN106075440A - A kind of hepatoma-targeting photo-thermal therapy agent and preparation method and application - Google Patents
A kind of hepatoma-targeting photo-thermal therapy agent and preparation method and application Download PDFInfo
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- CN106075440A CN106075440A CN201610457458.9A CN201610457458A CN106075440A CN 106075440 A CN106075440 A CN 106075440A CN 201610457458 A CN201610457458 A CN 201610457458A CN 106075440 A CN106075440 A CN 106075440A
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- biotinylation
- thermal therapy
- hepatoma
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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
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—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
- A61K49/0032—Methine dyes, e.g. cyanine dyes
- A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0058—Antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
Abstract
The invention provides a kind of hepatoma-targeting photo-thermal therapy agent and preparation method and application, belong to biomedical engineering field.The hepatoma-targeting photo-thermal therapy agent of the present invention is prepared by the following method and obtains: (1) prepares biotinylation nanometer microvesicle;(2) Cy7 fluorescently-labeled biotinylation resisting GPC 3 antibody is prepared;(3) biotinylation reproducibility graphene oxide (RGO) is prepared;(4) by biotin-labeled pentylamine system, the product coupling that above three step prepares is obtained hepatoma-targeting photo-thermal therapy agent.The photo-thermal therapy agent of the present invention has ultrasonic and fluorescence imaging dual-use function, technology mediation RGO targeting Delivery is destroyed by ultrasound targeted microbubble, monitoring photo-thermal therapy process in real time, this photo-thermal therapy agent can be used for the medicine of preparation treatment hepatocarcinoma, has the clinical value of excellence.
Description
Technical field
The present invention relates to biomedical engineering technology field, in particular it relates to a kind of hepatoma-targeting photo-thermal therapy agent and
Preparation method and application.
Background technology
Hepatocellular carcinoma (HCC) accounts for the 6th in world's cancer morbidity, and mortality rate accounts for the 3rd.Although hepatocarcinoma is with operation
Treatment is main, but the patient of only 9%~29% can excision.Photo-thermal therapy (Photothermal therapy, PTT)
It is a kind of Noninvasive thermal ablation therapy technology of development in recent years, mainly serves as photo-thermal having the material of light absorpting ability
Therapeutic agent, makes its local temperature raise killing tumor cell under laser irradiates.Tumor vessel is made up of two parts, a part
Being the intrinsic blood vessel of host, during tumor growth, the blood capillary major part of host local is destroyed, only tremulous pulse and relatively
Big vein may remain, and becomes the trunk of tumor vasculature, and this part vascular permeability is normal, the maximum of its tube wall
Aperture is less than 100nm;Another part is tumor neogenetic blood vessels, its structure imperfection, and basement membrane is imperfect, and tube wall is thin and lacks
Smooth muscle layer, permeability is significantly raised, and the maximum diameter of hole of its tube wall about 380~780nm, the most EV tumor cell can be straight
Connect and be connected with vessel lumen.Nanoscale microvesicle effectively can enter tumor tissues by the blood vessel wall in aperture about 380~780nm
Inside, more efficiently transhipment photo-thermal therapy agent.But general nanometer microvesicle is poor to the specificity of tumor tissues, it is impossible to ensure target
The photo-thermal therapy agent concentration that position is higher.
Preferably photo-thermal therapy agent should have stronger absorption, hypotoxicity, table at the reddest (outer light region 650-950nm)
Face can connect functional group and realize the active targeting treatment of tumor.PTT, can be real as a kind of Noninvasive thermal ablation techniques
The selective killing of existing cancerous cell, reduces normal cell and the damage of tissue.The photo-thermal of reproducibility graphene oxide (RGO) is controlled
Treatment curative effect affirmative, but RGO cannot independently image and without active targeting ability.RGO photo-thermal therapy is as a kind of potential wound
Little oncotherapy technology, gets the attention.But, need before the treatment to determine tumor by suitable imaging technique
The distribution in vivo situation of position, size and photo-thermal therapy agent and it is in the enrichment condition of tumor locus;Need shadow in the treatment
As monitoring tumor locus and the variations in temperature of surrounding normal tissue, to ensure good melting curative effect and avoid surrounding normal group
The damage knitted;PTT effect is evaluated after the treatment by imaging technique.Therefore, if can be to RGO photo-thermal therapy agent optimization liter
Level, obtains applying simultaneously to the Multifunction diagnosis and treatment integrated optical thermal therapeutical agent of ultrasonic-fluorescent dual module state imaging and treatment,
To realize the visualization photo-thermal therapy of tumor, it will be greatly improved the clinical therapeutic efficacy of hepatocarcinoma, alleviate patient suffering.
Summary of the invention
It is an object of the invention to provide a kind of can be applicable to ultrasonic fluorescence imaging hepatoma-targeting photo-thermal therapy agent and
Preparation method and application.
Present invention firstly provides the preparation method of a kind of hepatoma-targeting photo-thermal therapy agent, comprise the following steps:
(1) biotinylation nanometer microvesicle is prepared;
(2) Monophosphoinositideproteoglycans proteoglycans-3 of Cy7 fluorescently-labeled biotinylation anti-liver cancer and anti-cell membrane high expressed is prepared
(GPC3) antibody;
(3) biotinylation reproducibility graphene oxide is prepared;
(4) by biotin-avidin system, the product coupling that above three step prepares is obtained hepatoma-targeting photo-thermal
Therapeutic agent.
The present invention prepares the technical scheme of hepatoma-targeting photo-thermal therapy agent and sees Fig. 1.
In the said method of the present invention, it is by distearoyl phosphatidylcholine that step (1) prepares biotinylation nanometer microvesicle
(DSPC), DSPE-PEG (DSPE-PEG2000), biotinylation distearyl acyl group phosphatidyl
The mixture of ethanolamine-Polyethylene Glycol (DSPE-PEG2000-biotin) is dissolved in the mixed solution of chloroform and methanol, steams
Dry, add PBS and redissolve, be pipetted in centrifuge tube, seal, inner air is replaced into greenhouse gases;Vibration, water-bath,
Continue supersound process after the process of ultrasonic homogenizer, incubation, stand float assembly upper strata microsphere and be biotinylation nanometer microvesicle.
Further, described greenhouse gases are perfluoropropane (C3F8)。
Further, in step (1), the mixture of DSPC, DSPE-PEG2000, DSPE-PEG2000-biotin refers to
Three is 18:1:1 mixing according to mass ratio;The mixed solution of chloroform and methanol refers to that the volume ratio of the two is 2-2.5:1;Described
PBS pH value is 7.4, and concentration is 0.15mol/l;The temperature of described water-bath is 50-60 DEG C;Ultrasonic homogenizer after water-bath
The process time is less than 30 seconds;Heated culture temperature is 50-60 DEG C, and the time continuing supersound process is 1-2.5 minute.
Preferably, the mixture of DSPC, DSPE-PEG2000, DSPE-PEG2000-biotin refers to the mass ratio of three
For 18:1:1;The mixed solution of chloroform and methanol refers to that the volume ratio of the two is 2:1;Described PBS pH value is 7.4, dense
Degree is 0.15mol/l;The temperature of described water-bath is 55 DEG C;Ultrasonic homogenizer after water-bath processes 20 seconds time;Heated culture temperature is
55 DEG C, the time continuing supersound process is 2 minutes.
Step (1) uses ultrasonic homogenizer process to be conducive to making micron order microvesicle convert to nanoscale microvesicle, be conducive to
Reduce microvesicle particle diameter, and make the nanometer microvesicle in system more uniform, stable.In the inventive method, described in step (2)
Cy7 fluorescently-labeled biotinylation resisting GPC 3 antibody is prepared by the following method and obtains:
1. biotinylation resisting GPC 3 antibody is purchased from ABCAM company of Britain;
2. configuration cross-linking reaction liquid: accurately weigh NaHCO3Powder 0.378g, Na2CO3Powder 0.053g, NaCl powder
0.368g, in 50ml volumetric flask, adds aseptic double-distilled water and is settled to 50ml;
3. Cy7 powder is dissolved in DMSO, concentration 1mg/ml, omnidistance lucifuge operation;
4. antibody is dissolved in cross-linking reaction liquid with the concentration of 2mg/ml, is that 1mg is than 150 μ g by antibody and Cy7 mass ratio
Ratio reaction, Cy7 fluorescent dye is slowly added in antibody linked reactant liquor, jiggles mixing, lucifuge 4 DEG C reaction 8 is little
Time;
5. the NH of 5mol/L is dripped4Cl solution 25uL terminates reaction, and lucifuge 4 DEG C continues reaction 8 hours;
6. gained cross-linking reaction liquid is isolated and purified by sephadex G 10 chromatographic column lucifuge, and use freezer dryer will
Filtrate lucifuge lyophilizing, the Cy7 fluorescence-labeled bio element resisting GPC 3 antibody that obtained freeze-drying powder is the most prepared.
It is anti-that the present invention uses sephadex G 10 chromatographic column to be capable of the most isolated and purified Cy7 fluorescent labeling
GPC3 antibody, can obtain optimal isolated and purified effect compared with other chromatographic columns.
In the inventive method, the biotinylation reproducibility graphene oxide described in step (3) is prepared by the following method
Arrive, ultrasonic after biotinylation Polyethylene Glycol is mixed according to mass ratio 1:10 ratio with 2mg/mL reproducibility graphene oxide solution
Process 80-100min, be then centrifuged for removing unstable RGO, take supernatant, remove unnecessary biotinylation with membrane filtration and gather
Ethylene glycol, obtains the biotinylation reproducibility graphene oxide RGO-PEG-Biotin with biocompatibility.
Preferably, sonication treatment time is 90min.Using ultrasonic homogenizer, 20kHz carries out supersound process.
In the inventive method, the coupling method of step (4) comprises the following steps:
1. the biotinylation nanometer microbubble concentration that set-up procedure (1) prepares is 1 × 108-3×108/ ml), add excess
Streptavidin mixes, and reacts 30min, obtain solution 1 at 37 DEG C;
The most again the Cy7 fluorescently-labeled biotinylation resisting GPC 3 antibody that step (2) prepares is adjusted to concentration 1-1.5mg/
After ml, being added dropwise in solution 1, front and back the volume ratio of the two is 4:1, mixing, reacts 30min at 37 DEG C;Centrifugal, draw lower floor
Clear liquid, washes away unconjugated antibody and obtains solution 2;
The most then add in solution 2 excess biotinylation reproducibility graphene oxide (RGO-PEG-Biotin), 4 DEG C
Stand 30 minutes;
4. rinse with PBS, wash away unconjugated RGO-PEG-Biotin, formed and carry resisting GPC 3 antibody and RGO-PEG-
The nanoscale ultrasonic microbubble of Biotin, is hepatoma-targeting photo-thermal therapy agent.
The invention provides the hepatoma-targeting photo-thermal therapy agent that above-mentioned preparation method prepares.
Present invention also offers above-mentioned hepatoma-targeting photo-thermal therapy agent application in preparation treatment or diagnosing liver cancer medicine.
Further, the present invention provides a kind of medicine treating hepatocarcinoma, controls containing hepatoma-targeting photo-thermal of the present invention
Treat agent.
The photo-thermal therapy agent that the present invention prepares has ultrasonic and fluorescence imaging dual-use function, passes through ultrasound targeted microbubble
Destruction technology mediation RGO targeting Delivery, it is possible to monitoring photo-thermal therapy process in real time, can be used for the medicine of preparation treatment hepatocarcinoma, tool
There is the clinical value of excellence.
Accompanying drawing explanation
Fig. 1 is the method flow diagram that the present invention prepares hepatoma-targeting photo-thermal therapy agent.
Fig. 2 is the particle diameter test result of hepatoma-targeting photo-thermal therapy agent, and this result display complex average diameter is (316
± 31) nm, this complex particle diameter is normal distribution, dispersion 12.1%.
Fig. 3 is the picture of the hepatoma-targeting photo-thermal therapy agent that embodiment 4 prepares under optical microscope.
Fig. 4 is the picture that under laser confocal microscope, fluorescence is carried in the agent of hepatoma-targeting photo-thermal therapy.
Fig. 5 is confocal laser scanning microscope nanometer microvesicle complex and hepatoma carcinoma cell and the coupling picture of normal liver cell,
A, b figure is hepatoma carcinoma cell, and c, d figure is normal liver cell.
Fig. 6 is under optical microscope, and the HepG2 cell after hepatoma-targeting photo-thermal therapy agent treatment is thin with untreated HepG2
Born of the same parents scheme, a, b figure reflection fucosylation ability, c, d figure reflection Invasive Ability of Hepatocellular Carcinoma.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.Without departing substantially from present invention spirit
In the case of essence, the amendment that the inventive method, step or condition are made or replacement, belong to the scope of the present invention.
If not specializing, chemical reagent used in embodiment is conventional commercial reagent, skill used in embodiment
The conventional means that art means are well known to those skilled in the art.
Prepared by embodiment 1 biotinylation nanometer microvesicle (Nanobubble)
1. DSPC, DSPE-PEG2000, the DSPE-PEG2000-biotin selecting mass ratio to be 18:1:1 is raw material, will
10mg mixture of phospholipids is placed in 20ml round-bottomed flask, is added dropwise over chloroform and methanol mixed solution (volume ratio 2:1), until fat
Matter mixture is completely dissolved.Flask is placed in rotary evaporation instrument, and lower 40 DEG C of vacuum state is evaporated;
2., after being evaporated, flask adds 5ml PBS (pH=7.4,0.15mol/l) and redissolves, be pipetted into centrifugal
Guan Zhong, seals, inner air is replaced into C3F8;
3. argental mercury agitator shakes 40 seconds, then solution is placed in water-bath at temperature 55 DEG C, uses ultrasonic homogenizer (work simultaneously
Working frequency 20kHz) carry out processing 20 seconds.After process terminates, same temperature is hatched 60 minutes, continues ultrasonic homogenizer afterwards and processes
2 minutes.Stand float assembly upper strata microsphere the most made biotinylation nanometer microvesicle.
4. the form of electron microscope observation nanometer microvesicle and size, mean diameter (497.9 ± 30.9) nm.Sun under light microscopic
Ion microvesicle form is regular, and size is uniform, good dispersion degree.See Fig. 2.
The Cy7 fluorescent labeling of embodiment 2 biotinylation resisting GPC 3 antibody
1. biotinylation resisting GPC 3 antibody is purchased from ABCAM company of Britain;
2. configuration cross-linking reaction liquid: essence claims NaHCO3Powder 0.378g, Na2CO3Powder 0.053g, NaCl powder 0.368g
In 50ml volumetric flask, add aseptic double-distilled water and be settled to 50ml;
3. Cy7 powder is dissolved in DMSO, concentration 1mg/ml, omnidistance lucifuge operation;
4. antibody is dissolved in cross-linking reaction liquid with the concentration of 2mg/ml, is that 1mg compares 150ug by antibody and Cy7 mass ratio
Ratio reaction, Cy7 fluorescent dye is slowly added in antibody linked reactant liquor, jiggles mixing, lucifuge 4 DEG C reaction 8 is little
Time;
5. the NH of 5mol/L is dripped4Cl solution 25 μ L terminates, and lucifuge 4 DEG C continues reaction 8 hours;
6. gained cross-linking reaction liquid is isolated and purified by sephadex G 10 chromatographic column lucifuge, and use freezer dryer will
Filtrate lucifuge lyophilizing, the Cy7 fluorescence-labeled bio element resisting GPC 3 antibody that obtained freeze-drying powder is the most prepared.
The preparation of embodiment 3 biotinylation reproducibility graphene oxide
1. weigh 1g powdered graphite, add the NaCl powder mixing of about 40g, by ceramic mortar, this mixture is fully ground
Mill, to visible without obvious graphite granule.It is dissolved in water and filters and wash away NaCl powder in mixture, and finely ground graphite is placed in
Baking oven is dried.Powdered graphite after drying adds in 250mL round-bottomed flask, is carefully added into the dense sulfur of about 23mL while stirring
Acid, is stirred at room temperature 8 hours until concentrated sulphuric acid and graphite are sufficiently mixed uniformly.Reaction vessel is placed in ice-water bath, waits to burn
After mixture temperature drops to less than 15 DEG C in bottle, the most gradually add 3g potassium permanganate solid altogether, adding procedure is constantly stirred
Mix, it is ensured that during this, pot temperature keeps uniformly stablizing.After potassium permanganate solid adds, with heating mantle heats gradually
It is warming up to 75 DEG C, is incubated 45min, and continuously stirred.In reactant, now it is rapidly added about 3mL distilled water continuous stir about
5min, comes again rear question response thing variable color, heated up and can add 40mL distilled water and temperature rises to 100 DEG C after 100 DEG C, protects
15min is stirred under temperature.Backward reactant in add about 140mL distilled water, and add 10mL 30% hydrogenperoxide steam generator,
React after stirring 5min and terminate.It is placed in 50mL centrifuge tube after the cooling of question response thing, is retained precipitation so that 8000rpm is centrifugal
And wash respectively for several times with 5% dilute hydrochloric acid solution and distilled water, to solution solute can not again by 8000rpm be centrifuged for
Only, gained solution is graphene oxide colloid solution, stores for future use.
2. hydrazine hydrate reduction graphene oxide (GO) is utilized to prepare reproducibility graphene oxide.Hydrazine hydrate is pressed with GO solution
Mix according to 1:10 ratio, 95 DEG C of water-baths are reacted 24h, removes unnecessary hydrazine hydrate by Suction filtration device washing afterwards, obtain
The reproducibility graphene oxide (RGO) of solid, shaped.
3. due in reduction process RGO surface functional group be reduced effect remove, so RGO is water insoluble.In order to obtain
Water miscible RGO, uses biotinylation Polyethylene Glycol (PEG-Biotin) to obtain RGO-PEG-by non-covalent modification RGO
Biotin.PEG-Biotin with 2mg/mL RGO solution mix according to mass ratio 1:10 ratio after ultrasonic 90min.Then use
14800rpm is centrifuged 3h and removes unstable RGO, takes supernatant, removes unnecessary PEG-Biotin with the membrane filtration of 100nm,
Obtain that there is the RGO (RGO-PEG-Biotin) that the PEG-Biotin of biocompatibility modifies.
Embodiment 4 preparation carries fluorescence nano microvesicle complex (the hepatoma-targeting photo-thermal therapy of reproducibility graphene oxide
Agent)
1, the preparation of hepatoma-targeting photo-thermal therapy agent
1. (concentration is 3 × 10 to the biotinylation nanometer microvesicle 2ml that extraction embodiment 1 prepares8/ ml), add excess
Streptavidin, slight piping and druming makes it mix, and reacts 30min, obtain solution 1 at 37 DEG C;
The fluorescently-labeled biotinylation resisting GPC 3 antibody aseptic double-distilled water the most again embodiment 2 prepared adjust to
After concentration 1mg/ml, being added dropwise in solution 1, front and back the volume ratio of the two is 4:1, and slight piping and druming makes it mix, anti-at 37 DEG C
Answer 30min;Centrifugal, draw subnatant, wash away unconjugated antibody and obtain solution 2;
The most then adding the excessive RGO-PEG-Biotin that embodiment 3 prepares in solution 2,4 DEG C stand 30 minutes.
Rinse twice with PBS the most again, wash away unconjugated RGO-PEG-Biotin, formed and carry resisting GPC 3 antibody and RGO-
The nanoscale ultrasonic microbubble of PEG-Biotin, hepatoma-targeting photo-thermal therapy agent the most of the present invention, 4 DEG C keep in Dark Place, optics
Under microscope, Fig. 3 is shown in by picture.
2, the sign of the fluorescence nano microvesicle complex of redox graphene is carried
1. (what i.e. above-mentioned steps prepared carries resisting GPC 3 antibody and RGO-PEG-Biotin to take a small amount of microvesicle complex
Nanoscale ultrasonic microbubble) characterize form and the deployment conditions of nanometer microvesicle complex in optical microscope.Cytometry
Instrument calculates the concentration of nanometer microvesicle complex;Dynamic light scattering-laser particle analyzer and Zeta potential measuring instrument, measure its particle diameter and
Zeta potential, Fig. 4 shows that the RGO by CY7 red fluorescence labelling and the GPC3 antibody by green fluorescent label are tight with nanometer microvesicle
Close connected, it was demonstrated that to carry RGO nano target microvesicle complex and successfully construct.
2. the UV, visible light near infrared spectrometer complex to preparing absorption spectrum in water and in culture medium is used to enter
Row test, test deionized water is as reference substance, and scanning wavelength scope is 200-900nm.Result display complex is the reddest
At outer smooth region 808nm, there is stronger absorption.
The targeting detection of the hepatoma-targeting photo-thermal therapy agent of embodiment 5 present invention
1. In Culture Hepatoma Cell strain HepG2 and normal liver cell strain LO2: cell strain are containing 10% hyclone
In DMEM culture fluid, the CO of 5%2, 37 DEG C of descending regular growth subcultures, the cell of trophophase of taking the logarithm is tested.
2. targeting experiment: experiment point two groups: HepG2 hepatoma carcinoma cell group, LO2 normal liver cell matched group,
HepG2, LO2 Tissue Culture Plate is separately added into the hepatoma-targeting photo-thermal therapy agent that the embodiment 4 of equivalent prepares, hatches 2 for 37 DEG C
Hour, then wash 3 times with PBS liquid, finally use confocal laser scanning microscope nanometer microvesicle complex and hepatoma carcinoma cell and
The coupling situation of normal liver cell.Result as it is shown in figure 5, HepG2 hepatoma carcinoma cell group experimental result such as Fig. 5 a figure, b figure shown in,
Under laser confocal microscope, the hepatoma-targeting photo-thermal therapy agent of band green fluorescence is combined closely with hepatoma carcinoma cell, and PBS rinses not
Can be isolated, it was demonstrated that this therapeutic agent is tightly combined with hepatoma carcinoma cell targeting, there is good targeting ability;LO2 normal hepatocytes is thin
Born of the same parents' matched group experimental result such as schemes c, and shown in d, under laser confocal microscope, the most less green fluorescence is tied with normal liver cell
Close (or be hepatocellular normal endocytosis, cause this therapeutic agent to enter in cell cytoplasm), it was demonstrated that the hepatocarcinoma target of the present invention
To photo-thermal therapy agent, there is liver cancer-specific targeting binding ability.
The therapeutic effect detection of the hepatoma-targeting photo-thermal therapy agent of embodiment 6 present invention
1. fucosylation capacity experimental after treatment:
The hepatoma-targeting photo-thermal therapy agent that embodiment 4 prepares is added in HepG2 Tissue Culture Plate, near with 808nm afterwards
Infrared light carries out photo-thermal therapy.Take the HepG2 cell after treatment to test with untreated HepG2 cell.Respectively by 1
×104HepG2 before and after individual treatment is dissolved in 100 μ L serum-free DMEM complete culture solutions, is seeded in by 100 these Cell saps of μ L
The upper room (Costar company, the U.S.) of Transwell cell, the lower room at Transwell cell adds 800 μ L containing 10% afterwards
The DMEM complete culture solution of hyclone.Cultivate 12 hours in containing the CO2 of 5%, the incubator of 37 DEG C afterwards.Re-use 0.5%
The upper room lower wall of Transwell cell is dyeed by Crystal Violet Dye, the most again difference observation of cell number under optical microscope
Amount record.The a figure of result such as Fig. 6, shown in b figure, in cell number a the to be less than figure in b figure, display is through photo-thermal therapy
HepG2 cell is compared with untreated HepG2 cell, through the Leukopenia of Transwell cell, it was demonstrated that control through photo-thermal
The cell migration ability treated substantially weakens.
2. Invasive Ability of Hepatocellular Carcinoma experiment after treatment:
By the DMEM complete culture solution mixing of the matrigel (BD company, the U.S.) ratio with 1:7 with serum-free, in order to simulate
Cellular matrix, detects Invasive Ability of Hepatocellular Carcinoma.Respectively by 5 × 104Before and after individual treatment, HepG2 cell is dissolved in 200 μ L serum-frees
DMEM complete culture solution, the upper room (Costar of the Transwell cell after 200 this Cell sap of μ L inoculations are processed by matrigel
Company, the U.S.), the lower room at Transwell cell adds the 800 μ L DMEM complete culture solution containing 10% hyclone afterwards.
Cultivate 24 hours in containing the CO2 of 5%, the incubator of 37 DEG C afterwards.Re-use 0.5% Crystal Violet Dye to Transwell cell
Upper room lower wall dye, respectively observation of cell quantity record under optical microscope the most again.The c figure of result such as Fig. 6, d
Shown in figure, in cell number c be less than the figure in d figure, display through the HepG2 cell of photo-thermal therapy with untreated
HepG2 cell is compared, through the Leukopenia of the Transwell cell containing matrigel, it was demonstrated that the cell through photo-thermal therapy is invaded
Ability of attacking substantially weakens.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, on the premise of without departing from the technology of the present invention principle, it is also possible to make some improvements and modifications, these improvements and modifications
Also should be regarded as protection scope of the present invention.
Claims (10)
1. the preparation method of a hepatoma-targeting photo-thermal therapy agent, it is characterised in that comprise the following steps:
(1) biotinylation nanometer microvesicle is prepared;
(2) Cy7 fluorescently-labeled biotinylation resisting GPC 3 antibody is prepared;
(3) biotinylation reproducibility graphene oxide is prepared;
(4) by biotin-avidin system, the product coupling that above three step prepares is obtained hepatoma-targeting photo-thermal therapy
Agent.
2. preparation method as claimed in claim 1, it is characterised in that step (1) prepare biotinylation nanometer microvesicle be by
The mixture of DSPC, DSPE-PEG2000, DSPE-PEG2000-biotin is dissolved in the mixed solution of chloroform and methanol, steams
Dry, add PBS and redissolve, seal, inner air is replaced into greenhouse gases;Vibration, water-bath, ultrasonic homogenizer process,
Continue supersound process after incubation, stand float assembly upper strata microsphere and be biotinylation nanometer microvesicle.
3. preparation method as claimed in claim 2, it is characterised in that DSPC, DSPE-PEG2000, DSPE-PEG2000-
The mixture of biotin refer to by three according to mass ratio be 18:1:1 mixing;The mixed solution of chloroform and methanol refers to the two
Volume ratio is 2-2.5:1;Described PBS pH value is 7.4, and concentration is 0.15mol/l;The temperature of described water-bath is 50-60
℃;Ultrasonic homogenizer after water-bath processed the time less than 30 seconds;Heated culture temperature is 50-60 DEG C, continues the time of supersound process
For 1-2.5 minute.
4. preparation method as claimed in claim 2, it is characterised in that DSPC, DSPE-PEG2000, DSPE-PEG2000-
The mixture of biotin refers to that the mass ratio of three is 18:1:1;The mixed solution of chloroform and methanol refers to that the volume ratio of the two is
2:1;Described PBS pH value is 7.4, and concentration is 0.15mol/l;The temperature of described water-bath is 55 DEG C;Ultrasonic after water-bath
Homogenizer processes 20 seconds time;Heated culture temperature is 55 DEG C, and the time continuing supersound process is 2 minutes.
5. preparation method as claimed in claim 2, it is characterised in that the biotinylation reproducibility oxidation stone described in step (3)
Ink alkene is prepared by the following method and obtains, by biotinylation Polyethylene Glycol with 2mg/mL reproducibility graphene oxide solution according to matter
Measure supersound process 80-100min after mixing than 1:10 ratio, be then centrifuged for removing unstable RGO, take supernatant, use filter membrane mistake
Filter off except unnecessary biotinylation Polyethylene Glycol, obtain the biotinylation reproducibility graphene oxide RGO-with biocompatibility
PEG-Biotin。
6. preparation method as claimed in claim 5, it is characterised in that sonication treatment time is 90min.
7. the preparation method as described in claim 1-6 is arbitrary, it is characterised in that the coupling method of step (4) includes following step
Rapid:
1. the biotinylation nanometer microbubble concentration that set-up procedure (1) prepares is 1 × 108-3×108/ ml, adds the strepto-parent of excess
And element, mixing, react 30min at 37 DEG C, obtain solution 1;
The most again the Cy7 fluorescently-labeled biotinylation resisting GPC 3 antibody that step (2) prepares is adjusted to concentration 1-1.5mg/ml,
Being added dropwise in solution 1, front and back the volume ratio of the two is 4:1, mixing, reacts 30min at 37 DEG C;Centrifugal, draw subnatant,
Wash away unconjugated antibody and obtain solution 2;
The most then adding excess RGO-PEG-Biotin in solution 2,4 DEG C stand 30 minutes;
4. rinse with PBS, wash away unconjugated RGO-PEG-Biotin, formed and carry resisting GPC 3 antibody and RGO-PEG-Biotin
Nanoscale ultrasonic microbubble, be hepatoma-targeting photo-thermal therapy agent.
8. the hepatoma-targeting photo-thermal therapy agent that the arbitrary described preparation method of claim 1-7 prepares.
9. the application in preparation treatment or diagnosing liver cancer medicine of the hepatoma-targeting photo-thermal therapy agent described in claim 8.
10. the medicine treating hepatocarcinoma, it is characterised in that containing the hepatoma-targeting photo-thermal therapy agent described in claim 8.
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