CN105126123B - The preparation method of nano-probe, the preparation method and application of Nano medication based on natural products monomer and nano-probe - Google Patents
The preparation method of nano-probe, the preparation method and application of Nano medication based on natural products monomer and nano-probe Download PDFInfo
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Abstract
The present invention provides a kind of preparation method of nano-probe, the preparation method and application of Nano medication based on natural products monomer and nano-probe, the preparation method of the Nano medication is to pass through amidation process, natural products monomer and amination PEG are connected into drug molecule PEG polymer, then the polymer is connected to by quantum dot surface by the method for phase transfer, and then forms the fluorescence nano medicine with targeting.The present invention is by the method for simple possible by the surface of natural products monomer charge to nano-probe, it is oral from several formulations such as micro emulsion, solid lipid nano granule compared to conventional, the Nano medication of the micro-nano structure is more beneficial for the release of medicine, improve utilization ratio of drug, and after surface modification, the fluorescent characteristic of nano-probe does not influence.
Description
Technical field
The invention belongs to field of medicaments, and in particular to a kind of preparation method of targeted nano probe, based on natural products list
The preparation method and application of the Nano medication of body and nano-probe, using nano-probe GC-QDs as carrier, load natural products list
The targeted nano medicine with photoluminescent property is prepared into after body, diagnostic flag and treatment available for hepatocellular carcinoma.
Background technology
Hepatocellular carcinoma（Hepatocellular carcinoma, HCC）Be by virus and chemical carcinogen etc. it is more because
The effect of element causes hepatic cell growth out of control and carcinogenesis.HCC grade malignancies are high, and prognosis is very poor, and it is to improve HCC that early diagnosis, which is early controlled,
The unique channel of survival.GPC-3 expression and the formation of hepatocellular carcinoma are in close relations, have cancer embryo, can be used as early stage
HCC blood serum designated object and potential targeted therapy target.By establishing high specific and highly sensitive GPC-3 monitoring sides
Method, it can be expected to realize targeting HCC Accurate Diagnosis and treatment in time.
Celastrol is a kind of natural activity monomer with notable antitumor activity.Because the poison of Celastrol is secondary
Effect is also relatively large, and therefore, the transformation of structure optimization and formulation to Celastrol is obtained with high-efficiency low-toxicity spy
The important channel of the antitumoral compounds of property.On the research of Celastrol novel form, report is few at present, mainly including mouth
Clothes prepare the research of Nano medication not from several formulations such as micro emulsion and solid lipid nano granule by way of area load
It is more.
Quantum dot can be used as a kind of preferably fluorescence probe and nano carrier material, diagnosis available for disease, control
The research field such as treatment and medicament research and development.But the cytotoxicity of conventional quantum dot is larger, biological safety and biological target tropism compared with
Difference.By structure of modification and functional modification, the shortcomings of quantum dot can be significantly improved, and then are applied to biomedicine
Research field.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of preparation method of nano-probe, based on natural products monomer and
The preparation method and application of the Nano medication of nano-probe, the Nano medication of preparation are more beneficial for the release of medicine, improve medicine
Utilization rate, and after surface modification, the fluorescent characteristic of nano-probe does not influence.
In order to solve the above technical problems, embodiments of the invention provide a kind of preparation method of nano-probe, the nanometer
Fluorescence quantum of the probe using ZnS as the core shell structure of shell is carrier, is prepared by surface modification monoclonal antibody GC-33, its
Preparation process is as follows：
（1-1）Prepare the nuclear shell structure quantum point using ZnS as shell；
（1-2）Quantum dot solution with core-shell structure of 2 ~ 10 ml using ZnS as shell is taken to have children outside the state plan scattered, 15000 rpm leave the heart
30 ~ 50 min, add ultra-pure water 0.5 ml, it is standby；
（1-3）5 ~ 20 mg carbodiimides is taken, 1 ~ 10mg N- hydroxy thiosuccinimides add the ml of water 1.0 to dissolve；
（1-4）The above-mentioned ml of solution 0.3 ~ 1.0 is taken to add using ZnS in the quantum dot solution with core-shell structure of shell, to be settled to
1.0 ~ 5.0 ml, 37 DEG C of shaking tables react 1 ~ 2 hour；
（1-5）15000 rpm high speed centrifugations 30 ~ 50 minutes, abandon supernatant, with the pH 8.5 μ l of PBS solution 250 ~ 1000 weights
New dissolving；
（1-6）Add 5 ~ 20 μ l targeting GPC-3 monoclonal antibody GC-33 solution, under 37 DEG C of constant temperature, shaking table reaction 2 ~ 3 is small
When, by monoclonal antibody GC-33 modifications to the surface using ZnS as the nuclear shell structure quantum point of shell；
（1-7）15000 rpm are centrifuged 30 ~ 50 minutes, are dissolved in pH 7.2 PBS solution, and it is swollen that separating-purifying obtains targeting
Tumor markers GPC-3 fluorescent nano probe.
Wherein, the step（1-1）In the nuclear shell structure quantum point using ZnS as shell be ZnO/ZnS, it is specific to prepare step
It is rapid as follows：
（2-1）3 ~ 8g zinc acetates and 0.3 ~ 1.5g sodium acetates are added in the round-bottomed flask equipped with 200mL ethanol；
（2-2）After heating for dissolving, the 1.0 mol/l mL of oxidation sodium ethoxide solution 18 ~ 50 is rapidly added, reacts 30 ~ 60 points
Clock, it is cooled to room temperature；
（2-3）In the solution with the fast Excess ethyl acetate that is added dropwise of 0.5 ~ 2.0 drop/sec of drop to there is precipitation to produce, centrifugation divides
Sediment is separated out, is washed 3 ~ 5 times with ethyl acetate, then sediment is disperseed again in ethanol, ZnO nano particle to be made；
（2-4）The 0.2 mol/l ml of zinc acetate 3 ~ 10 and isometric is added dropwise successively into the suspension of ZnO nano particle
0.2 mol/l thioacetamide ethanol solution, drip speed keep 0.5 ~ 2.0 drop/sec, heated after being added dropwise to complete；
（2-5）ZnS shell claddings are successively carried out to it according to S-Zn-S-Zn, form ZnO/ZnS quantum dots, reaction 4 ~ 8 is small
When after terminate, reuse the ZnO/ZnS core shell nanoparticles that acetic acid ethyl ester is settled out different-grain diameter, centrifuge out ZnO/
ZnS core-shell structured quantum dot.
The present invention also provides a kind of preparation method of the Nano medication based on natural products monomer and nano-probe, passes through acyl
Aminating reaction, natural products monomer and amination PEG are connected into drug molecule-PEG polymer, then pass through phase transfer
The polymer is connected to quantum dot surface by method, and then forms the fluorescence nano medicine with targeting.
The preparation method of above-mentioned Nano medication comprises the following steps：
（3-1）Nuclear shell structure quantum point using ZnS as shell is carrier, is prepared and targetted by surface modification monoclonal antibody GC-33
The tumor markers GPC-3 ml of fluorescent nano probe GC-QDs 1 ~ 10；
（3-2）Natural products monomer is taken to be configured to 1.0 ~ 10.0 mg/ml drug solution, it is standby；
（3-3）5 ~ 15mg carbodiimides, 1 ~ 5 mg N- hydroxy thiosuccinimides is taken to add 1.0 ~ 5.0ml of water molten
Solution；
（3-4）Take step（3-3）The made ml of solution 0.5, is added to 0.3 ~ 1.0ml by step（3-2）The medicine of configuration is molten
In liquid, 1.0 ml are settled to, 37 DEG C of shaking tables react 1 ~ 3 hour；
（3-5）Into above-mentioned mixed solution with 1 ~ 2 drop/sec of dropwise addition 2mg/ml amination PEG solution 1 ~ 5ml, Ran Hou
Reacted 2 ~ 4 hours under 37 DEG C of constant temperature, 200 ~ 1000 turns/min of mixing speed, be prepared into drug molecule-PEG polymer, it is pure
Dissolved again with PBS after change, it is standby；
（3-6）By drug molecule-PEG polymer solution and step（3-1）The nano-probe GC-QDs solution of configuration, is pressed
According to etc. mass concentration mixing, stirring reaction 3 ~ 6 hours at room temperature, drug molecule is connected to the surface of nano-probe；
（3-7）15000 rpm are centrifuged 30 ~ 50 minutes, collect supernatant, and sediment is redissolved in PBS, and target is made
To GPC-3 fluorescence nano medicine GC-QDs-Drug.
Wherein, the step（3-1）Middle nano-probe GC-QDs preparation process is with reference to noted earlier.
The present invention also provides a kind of application of above-mentioned Nano medication, including the external drug controlled release curve of Nano medication
Measure, Nano medication liver cancer cells are promoted to the measure of the Growth of Human Hepatoma Cell Line HepG 2 inhibiting rate of in vitro culture, Nano medication
The testing in vitro experiment of HepG2 apoptosis, Nano medication are to the marks of liver cancer cells.
The above-mentioned technical proposal of the present invention has the beneficial effect that：
1. the present invention by the method for simple possible by the surface of natural products monomer charge to nano-probe, compared to
It is conventional oral to be more beneficial for medicine from several formulations such as micro emulsion, solid lipid nano granule, the Nano medication of the micro-nano structure
Release, utilization ratio of drug is improved, and after surface modification, the fluorescent characteristic of nano-probe does not influence.
2. the fluorescence nano medicine prepared by the present invention has higher carrying drug ratio（20.31±6.76%）And envelop rate
（82.85±10.33%）, and pH sensitiveness is presented in vitro drug release, the environment compared to pH7.4, is situated between in pH6.0 acidity
In matter, Nano medication has faster drug releasing rate and drug release rate, and this characteristic reduces Nano medication and followed in body
Release amount of medicine and toxic side effect in ring, while be advantageous to the passive target effect in tumor locus.
3. the targeting fluorescence nano medicine prepared by the present invention can carry out fluorescence labeling and imaging to liver cancer cells, pass through
Integrated dynamic monitoring to the mark of cancer cell, tracer and treatment, development are referred to based on Novel marker GPC-3 marks and imaging
Lead the accurate treatment of lower liver cancer.
Brief description of the drawings
Fig. 1 be the present invention fluorescence nano medicine in the medium of different pH value, to the external medicine of Celastrol molecule
Thing release profiles；
Fig. 2 is the DAPI coloration results that liver cancer cells produce obvious apoptosis by Nano medication intervention afterwards in the present invention；
Fig. 3 be the present invention nano-probe and fluorescence nano medicine to the external fluorescence labeling experimental results of liver cancer cells
Figure.
Embodiment
For the ease of understanding the present invention, with reference to embodiment and accompanying drawing, the invention will be further described, but the present invention
Protection domain be not limited to embodiment in detail below.
Unless otherwise defined, the implication that all technical terms used hereinafter are generally understood that with those skilled in the art
Identical, technical term used in the present invention is intended merely to describe the purpose of specific embodiment, is not intended to the limitation present invention
Protection domain.
Unless otherwise specified, various raw material used in the present invention, reagent, instrument and equipment etc. can pass through
Market is commercially available or class is prepared by existing method.
Embodiment 1：
A kind of preparation method of nano-probe, fluorescence quantum of the nano-probe using ZnS as the core shell structure of shell
For carrier, it is prepared by surface modification monoclonal antibody GC-33, its preparation process is as follows：
（101-1）Prepare the nuclear shell structure quantum point using ZnS as shell；
Wherein, the nuclear shell structure quantum point using ZnS as shell is ZnO/ZnS, and its preparation method comprises the following steps：
（101-1-1）3.5g zinc acetates and 0.4g sodium acetates are added in the round-bottomed flask equipped with 200mL ethanol；
（101-1-2）After heating for dissolving, the 1.0 mol/l mL of oxidation sodium ethoxide solution 19.5 is rapidly added, reacts 30 points
Clock, it is cooled to room temperature；
（101-1-3）Excess ethyl acetate is added dropwise to there is precipitation to produce with 1 drop/sec of drop speed in the solution, centrifuged
Go out sediment, washed 3 ~ 5 times with ethyl acetate, then sediment is disperseed again in ethanol, ZnO nano particle to be made；
（101-1-4）0.2 the mol/l ml of zinc acetate 3.5 and grade body is added dropwise successively into the suspension of ZnO nano particle
The ethanol solution of the thioacetamide of 0.2 long-pending mol/l, drip speed and kept for 1 drop/sec, heated after being added dropwise to complete；
（101-1-5）ZnS shell claddings are successively carried out to it according to S-Zn-S-Zn, form ZnO/ZnS quantum dots, reaction 5
Terminated after hour, reuse the ZnO/ZnS core shell nanoparticles that acetic acid ethyl ester is settled out different-grain diameter, centrifuge out ZnO/
ZnS core-shell structured quantum dot.
（101-2）Take the 5.0 ml quantum dot solution with core-shell structure using ZnS as shell to have children outside the state plan to disperse, 15000 rpm turn
30 min are centrifuged, add ultra-pure water 0.5 ml, it is standby；
（101-3）10mg carbodiimides is taken, 2 mg N- hydroxy thiosuccinimides add the ml of water 1.0 to dissolve；
（101-4）The above-mentioned ml of solution 0.5 is taken to add using ZnS in the quantum dot solution with core-shell structure of shell, to be settled to
1.0 ml, 37 DEG C of shaking tables react 1 ~ 2 hour；
（101-5）15000 rpm high speed centrifugations 30 minutes, abandon supernatant, are dissolved again with the pH 8.5 μ l of PBS solution 250；
（101-6）Adding 5 μ l targeting GPC-3 monoclonal antibody GC-33 solution, under 37 DEG C of constant temperature, shaking table reacts 2 hours,
By monoclonal antibody GC-33 modifications to the surface using ZnS as the nuclear shell structure quantum point of shell；
（101-7）15000 rpm are centrifuged 30 minutes, are dissolved in pH 7.2 PBS solution, separating-purifying obtains target tumor
Mark GPC-3 fluorescent nano probe.
Embodiment 2：
A kind of preparation method of the Nano medication based on natural products monomer and nano-probe, will by amidation process
Natural products monomer connects into drug molecule-PEG polymer with amination PEG, is then gathered this by the method for phase transfer
Compound is connected to quantum dot surface, and then forms the fluorescence nano medicine with targeting.
Wherein, the natural products monomer in the present invention is from the natural activity monomer for possessing drug action, preferably molecule knot
Structure is with carboxyl and is the natural activity monomer of nonactive group, in the present embodiment so that Celastrol is drug molecule as an example, enters
One step describes the preparation method of Nano medication.
Comprise the following steps that：
（102-1）Nano-probe GC-QDs is prepared by embodiment 1；
（102-2）Celastrol is taken to be configured to 2.0mg/ml drug solution, it is standby；
（102-3）10mg carbodiimides EDC, 2 mg N- hydroxy thiosuccinimides NHSS is taken to add water 1.0ml molten
Solution；
（102-4）Take step（102-3）The made ml of solution 0.5, is added to 0.5ml by step（102-2）The medicine of configuration
In solution, 1.0 ml are settled to, 37 DEG C of shaking tables react 1 hour；
（102-5）Into above-mentioned mixed solution with 2 drops/sec of dropwise addition 2mg/ml amination PEG solution 1ml, then 37
DEG C constant temperature under hybrid reaction 2 hours, be prepared into drug molecule-PEG polymer, dissolved again with PBS after purification, it is standby；
（102-6）By drug molecule-PEG polymer solution and step（102-1）The nano-probe GC-QDs of configuration is molten
Liquid according to etc. mass concentration mix, stirring reaction 4 hours at room temperature, drug molecule is connected to the surface of nano-probe；
（102-7）15000 rpm are centrifuged 30 minutes, collect supernatant, and sediment is redissolved in PBS, and target is made
To GPC-3 fluorescence nano medicine GC-QDs-Drug.
The application of the Nano medication of above-mentioned preparation, include measure, the nanometer of the external drug controlled release curve of Nano medication
Medicine promotes hepatocellular carcinoma H22 apoptosis to the measure of the Growth of Human Hepatoma Cell Line HepG 2 inhibiting rate of in vitro culture, Nano medication
Testing in vitro experiment, Nano medication are to the marks of liver cancer cells.
（One）The measure of the external drug controlled release curve of Nano medication, step are as follows：
（103-1）The fluorescence nano medicine of preparation is dissolved in 5 mL PBS cushioning liquid, and is transferred to bag filter
In；
（103-2）Bag filter is placed in and fills 95 mL, pH 7.4 in cushioning liquid, 100 rpm in 37 DEG C of waters bath with thermostatic control
At the uniform velocity stir buffer solution；
（103-3）Respectively at the mL of 0,2,4,6,8,10,12,16,20,24 h timing samplings 0.2, while cover same volume
Corresponding cushioning liquid；
（103-4）The sample of resulting different time points detects the concentration of Celastrol with high-efficient liquid phase technique；Chromatogram
Condition is：Chromatographic column：Dikma Technologies C18（250 mm × 4. 6 mm, 5 μm）, mobile phase is methanol：10
ML/L acetums（87：13）；Detection wavelength：425 nm；Column temperature：25 ℃；Flow velocity：1.0 mL/min；Sample size: 20 μL；
（103-5）The amount of the Celastrol of Nano medication GC-QDs-Drug releases when calculating different time points, then calculate
Cumulative release amount simultaneously draws In-vitro release curves；With cumulative release amount and time match equation, if trypterygine in CSL-CdTe
Plain CSL quality is Mdrug, and release cushioning liquid PBS volume is V0（That is 100 mL）, PBS displaced volume is Vs（I.e.
0.2 mL）, the sample drug concentration that the i-th moment took out is Ci, with the cumulative release of following formula calculating Celastrol
Amount：
。
（103-6）In the media environment that pH of buffer 6.0 is measured under same steps, medicine of the Nano medication to drug molecule
Thing release profiles；
（103-7）Accompanying drawing 1 is that drug accumulation release percentage changes over time curve under the conditions of pH 6.0 and pH 7.4.
From curve：The release of drug molecule has faster release in the incipient stage in prepared Nano medication
Speed, afterwards rate of release tend towards stability.And the release rate of drug molecule has correlation with residing pH environment, present bright
Aobvious pH sensitiveness.As a result show, in pH7.4 dissolution medium, after a faster drug release phase, release
Speed gradually tends to be slow, and preparation is 30.9% in 24 hours.And in pH 6.0 sour environment, medicine within preceding 8 h
Thing molecule is respectively provided with faster drug releasing rate and drug release rate, and 8 h drug release rate is 56.4%, is tired out in 24 hours
Product release rate reaches 86.5%.
（Two）Nano medication is as follows to the measure of the Growth of Human Hepatoma Cell Line HepG 2 inhibiting rate of in vitro culture, step：
（104-1）Trypsin Induced is in the hepatocellular carcinoma H22 of exponential phase, and density is made as 1 × 105Individual/
ML single cell suspensions, it is inoculated in 96 orifice plates；
（104-2）Cell is in 37 DEG C, contains 5% CO2Humidified incubator in continue cultivate 24 h after change fresh training
Nutrient solution, it is respectively 0.0625 μ g/mL, 0.125 μ g/mL, 0.25 μ g/mL, 0.50 μ g/mL, 1.00 μ g/mL, 2.00 μ with concentration
G/mL Celastrol solution intervenes liver cancer cells, as negative control group；With the nanometer equivalent to Celastrol concentration
Medicine GC-QDs-Drug intervenes hepatocellular carcinoma H22 and positive group of conduct；And set up blank control group；
（104-3）Cell continues to cultivate 24 h, 48 h and 72 h respectively in incubator, and each concentration point sets three again
Hole；
（104-4）Incubation time adds 5 mg/mL tetrazolium bromide per hole after terminating（MTT）The μ L of solution 20 continue culture 4
h；
（104-5）Supernatant is sucked, dimethyl sulfoxide (DMSO) is added per hole（DMSO）150 μ L, terminating reaction；
（104-6）96 orifice plates are moved into plate shaker, lucifuge 10 min of horizontal concussion, make MTT reduzates completely molten
Solution, then determined with enzyme-linked immunosorbent assay instrument at 492 nm wavelength per hole absorbance OD values, and with the OD values of blank control wells
Zeroing, is as a result represented with the mean ± standard error of every group of 3 multiple holes, and experiment is at least repeated 3 times；
Inhibitory rate of cell growth is calculated with following formula：
Inhibitory rate of cell growth（%）=[1- (OD experiment values/OD control values)] × 100%.
（104-7）The result such as table 1 that each medicine group detects to the MTT of the hepatocellular carcinoma H22 proliferative effect of in vitro culture
It is shown.When simple Celastrol intervenes cell, there is certain cytotoxicity to hepatocellular carcinoma H22, and cytotoxicity is in
Existing dose dependent and time dependence.When Nano medication GC-QDs-Drug is added into cell, relative to check experiment, carefully
The inhibiting rate of born of the same parents substantially increases, after intervening culture 24h, 48h and 72h, its IC50Value be reduced to respectively 2.90 μ g/mL,
0.90 μ g/mL and 0.34 μ g/mL, hence it is evident that less than the IC of control group experiment50As a result 14.20 μ g/mL, 3.91 μ g/mL and
1.47 μg/mL。
Table 1：
According to above-mentioned IC50The results contrast of value can be seen that targeted nano medicine GC-QDs-Drug and clearly enhance thunder
Celastrol molecule improves the drug effect of drug molecule to the toxicity of hepatocellular carcinoma H22.
（Three）Nano medication promotes the testing in vitro experiment of hepatocellular carcinoma H22 apoptosis, comprises the following steps：
（105-1）Trypsin Induced is in the hepatocellular carcinoma H22 of exponential phase, and it is 1 × 105/mL that density, which is made,
Single cell suspension, it is inoculated in 96 orifice plates；
（105-2）Cell is in 37 DEG C, contains 5% CO2Humidified incubator in continue cultivate 24 h after change fresh training
Nutrient solution, it is incubated altogether with hepatocellular carcinoma H22 with 1 μ g/mL Celastrol and Nano medication GC-QDs-Drug respectively；
（105-3）It is incubated after 24 h, discards culture medium, after pH 7.4 PBS is rinsed 2 ~ 3 times, methanol is fixed
10min；
（105-4）Methanol is discarded, after drying, adds DAPI working solutions to wash 10 min of dyeing；
（105-5）Dyeing liquor is discarded, background is rinsed 2 ~ 3 times with the PBS solution of pH value 7.4, then with distilled water flushing, is dried
After observe, fluorescence microscope is observed, taken pictures with 340/380 nm ultraviolet excitations；
（105-6）Fig. 2 is the coloration result that each medicine group promotes liver cancer HepG2 apoptosis
As seen from the figure：Blank group without pharmaceutical intervention（A groups）Cell be colored after, intracellular fluorescence presents equal
Even distribution.When cell passes through 1.0 μ g/mL Celastrols（B groups）After intervention, it is observed that double-stranded DNA in nucleus
Fluorescence intensity be remarkably reinforced, in appearance that is crescent, and having a small amount of apoptotic body.When cell passes through the nanometer medicine of the same terms
Thing GC-QDs-Drug（C groups）After intervention, it is observed that chromatin concentration, the appearance of a large amount of apoptotic bodies in nucleus
Deng.As a result prove, nano combined medicine significantly enhances the cytotoxicity of drug molecule, promotes a large amount of of liver cancer cells and withers
Die.
（Four）Laser confocal fluorescence microscope studies fluorescence nano medicine GC-QDs-Drug to hepatocellular carcinoma H22
Mark, step are as follows：
（106-1）Trypsin Induced is in the hepatocellular carcinoma H22 of exponential phase, and it is 1 × 105/mL that density, which is made,
Single cell suspension, it is inoculated in 35 mm thin layer glass bottom（The mm of 0.13 ~ 0.16 mm thick, 20 mm × 20）In culture dish；
（106-2）Cultivate 24 h and change fresh medium afterwards, cell is respectively with 1 μ g/mL Celastrol, 5 μ g/
ML nano-probe GC-QDs and 5 μ g/mL Nano medication GC-QDs-Drug carry out common incubation；
（106-3）Culture medium is discarded after being incubated 6 h, cell is gently washed 2 times with the PBS solution of pH value 7.4, finally will
Cell is placed in PBS environment；
（106-4）Culture dish is placed directly within laser confocal fluorescence microscope and observed, is taken pictures.Laser co-focusing fluorescence
Microscopic system passes through rotating circular disk laser system（Revolution XD, Andor Technology, Northern
Ireland）Cause fluorescence microscope with Ti-E（Nikon, Japan）Assemble；Confocal Images pass through electron multiplication CCD
（EMCCD）iXon DV885（Andor）Obtained with 1004 × 1002 pixels, scan size is 177.3 × 177.5 μm.It is logical
Cross acousto-optic turnable filter（AOTF）405 nm solid state fluorescences excitation quantum points are modulated, use 585/40 transmitting grating
（Semrock, USA）, 60x oil mirrors（Apo, NA.=1.49, Nikon）Observe cell.The image of gained is v.1.10 soft by iQ
Part processing（Andor）, as a result see Fig. 3.
（106-5）Liver cancer cells nano-probe GC-QDs or nano combined medicine GC-QDs-Drug are incubated it jointly
Afterwards after 6 hours, to liver cancer cells mark, the laser co-focusing experimental result being imaged as shown in figure 3, passing through Laser Scanning Confocal Microscope
Different liver cancer cells are can clearly be observed that, fluorescence labeling is carried out to cell.Marked with simple nano-probe GC-QDs（B groups）
Result compare, Celastrol molecule be supported on nano-probe surface composition Nano medication GC-QDs-Drug do not have shadow
Ring the photoluminescent property to nano-probe（C groups）, while Celastrol intervention group under the experimental condition（A groups）It is not fluoresce
, therefore, new fluorescence nano medicine can be applied to the mark of liver cancer cells as a kind of targeting fluorescent probe of multi-mode
In note, imaging, by the mark to tumor markers GPC-3, tracer, it is expected to realize the accurate treatment that liver cancer occurs, develops.
The present invention by the method for simple possible by the surface of natural products monomer charge to nano-probe, compared to commonly using
It is oral be more beneficial for the release of medicine from several formulations such as micro emulsion, solid lipid nano granule, the Nano medication of the micro-nano structure,
Utilization ratio of drug is improved, and after surface modification, the fluorescent characteristic of nano-probe does not influence.
Described above is the preferred embodiment of the present invention, it is noted that for those skilled in the art
For, on the premise of principle of the present invention is not departed from, some improvements and modifications can also be made, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (6)
1. a kind of preparation method of the Nano medication based on natural products monomer and nano-probe, it is characterised in that pass through acid amides
Change reaction, connect by molecular structure with carboxyl and for nonactive group, tool drug action natural products monomer and amination PEG
It is connected into drug molecule-PEG polymer；Nuclear shell structure quantum point using ZnS as shell is carrier, passes through surface modification monoclonal antibody
GC-33 prepares target tumor mark GPC-3 fluorescent nano probe GC-QDs；Then by by drug molecule-PEG polymerization
Thing solution and nano-probe GC-QDs solution according to etc. mass concentration mix the method for making drug molecule by phase transfer by medicine
Molecule is connected to fluorescent nano probe GC-QDs surface, and then forms targeting GPC-3 fluorescence nano medicine GC-QDs-
Drug。
2. the preparation method of Nano medication according to claim 1, it is characterised in that comprise the following steps：
（3-1）Nuclear shell structure quantum point using ZnS as shell is carrier, and target tumor is prepared by surface modification monoclonal antibody GC-33
The mark GPC-3 ml of fluorescent nano probe GC-QDs 1 ~ 10；
（3-2）Natural products monomer is taken to be configured to 1.0 ~ 10.0 mg/ml drug solution, it is standby；
（3-3）5 ~ 15mg carbodiimides, 1 ~ 5 mg N- hydroxy thiosuccinimides is taken to add 1.0 ~ 5.0ml of water to dissolve；
（3-4）Take step（3-3）The made ml of solution 0.5, is added to 0.3 ~ 1.0ml by step（3-2）The drug solution of configuration
In, 1.0 ml are settled to, 37 DEG C of shaking tables react 1 ~ 3 hour；
（3-5）Into above-mentioned mixed solution with 1 ~ 2 drop/sec of dropwise addition 2mg/ml amination PEG 1 ~ 5ml of solution, then at 37 DEG C
Constant temperature under react 2 ~ 4 hours, 200 ~ 1000 turns/min of mixing speed, be prepared into drug molecule-PEG polymer, after purification
Dissolved again with PBS, it is standby；
（3-6）By drug molecule-PEG polymer solution and step（3-1）The nano-probe GC-QDs solution of configuration, according to etc.
Mass concentration is mixed, at room temperature stirring reaction 3 ~ 6 hours, and drug molecule is connected to the surface of nano-probe；
（3-7）15000 rpm are centrifuged 30 ~ 50 minutes, collect supernatant, and sediment is redissolved in PBS, and targeting is made
GPC-3 fluorescence nano medicine GC-QDs-Drug.
3. the preparation method of Nano medication according to claim 2, it is characterised in that the step（3-1）Middle nanometer is visited
Pin GC-QDs preparation process is as follows：
（4-1）Prepare the nuclear shell structure quantum point using ZnS as shell；
（4-2）Take quantum dot solution with core-shell structure of 2 ~ 10 ml using ZnS as shell to have children outside the state plan scattered, 15000 rpm leave the heart 30 ~
50 min, add ultra-pure water 0.5 ml, it is standby；
（4-3）5 ~ 20 mg carbodiimides is taken, 1 ~ 10mg N- hydroxy thiosuccinimides add the ml of water 1.0 to dissolve；
（4-4）Take the above-mentioned ml of solution 0.3 ~ 1.0 add using ZnS as in the quantum dot solution with core-shell structure of shell, be settled to 1.0 ~
5.0 ml, 37 DEG C of shaking tables react 1 ~ 2 hour；
（4-5）15000 rpm high speed centrifugations 30 ~ 50 minutes, abandon supernatant, again molten with the pH 8.5 μ l of PBS solution 250 ~ 1000
Solution；
（4-6）Add 5 ~ 20 μ l targeting GPC-3 monoclonal antibody GC-33 solution, under 37 DEG C of constant temperature, shaking table reacts 2 ~ 3 hours, will
The surface using ZnS as the nuclear shell structure quantum point of shell is arrived in monoclonal antibody GC-33 modifications；
（4-7）15000 rpm are centrifuged 30 ~ 50 minutes, are dissolved in pH 7.2 PBS solution, separating-purifying obtains target tumor mark
Will thing GPC-3 fluorescent nano probe.
4. the application of a kind of Nano medication as prepared by any one of claim 1 ~ 3, it is characterised in that including Nano medication
The measure of external drug controlled release curve, Nano medication to the measure of the Growth of Human Hepatoma Cell Line HepG 2 inhibiting rate of in vitro culture,
Nano medication promotes the testing in vitro experiment of hepatocellular carcinoma H22 apoptosis.
5. the application of Nano medication according to claim 4, it is characterised in that Nano medication is thin to the liver cancer of in vitro culture
The measure of born of the same parents' HepG2 growth inhibition ratios, step are as follows：
（5-1）Trypsin Induced is in the hepatocellular carcinoma H22 of exponential phase, and density is made as 1 × 105Individual/mL is slender
Born of the same parents' suspension, it is inoculated in 96 orifice plates；
（5-2）Cell is in 37 DEG C, contains 5% CO2Humidified incubator in continue cultivate 24 h after change fresh medium, use
Concentration is respectively 0.0625 μ g/mL, 0.125 μ g/mL, 0.25 μ g/mL, 0.50 μ g/mL, 1.00 μ g/mL, 2.00 μ g/mL medicine
Thing molecular solution intervenes liver cancer cells, as negative control group；With the Nano medication intervention equivalent to drug molecule solution concentration
Liver cancer cells and positive group of conduct；And blank control group is set up,
（5-3）Cell continues to cultivate 24 h, 48 h and 72 h respectively in incubator, and each concentration point sets three multiple holes；
（5-4）Incubation time adds 5 mg/mL per the hole μ L of tetrazolium bromide MTT solution 20 after terminating continue to cultivate 4 h；
（5-5）Supernatant is sucked, the μ L of dimethyl sulfoxide (DMSO) 150, terminating reaction are added per hole；
（5-6）96 orifice plates are moved into plate shaker, lucifuge 10 min of horizontal concussion, are completely dissolved MTT reduzates, then
Determined per hole absorbance OD values at 492 nm wavelength with enzyme-linked immunosorbent assay instrument, and returned to zero with the OD values of blank control wells, knot
Fruit represents that experiment is at least repeated 3 times with the mean ± standard error of every group of 3 multiple holes；
Inhibitory rate of cell growth is calculated with following formula：
Inhibitory rate of cell growth（%）=[1- (OD experiment values/OD control values)] × 100%.
6. the application of Nano medication according to claim 4, it is characterised in that Nano medication promotes hepatocellular carcinoma H22
The testing in vitro experiment of apoptosis, comprises the following steps：
（6-1）Trypsin Induced is in the hepatocellular carcinoma H22 of exponential phase, and density is made as 1 × 105Individual/mL is slender
Born of the same parents' suspension, it is inoculated in 96 orifice plates；
（6-2）Cell is in 37 DEG C, contains 5% CO2Humidified incubator in continue cultivate 24 h after change fresh medium, point
It is not incubated altogether with hepatocellular carcinoma H22 with 1 μ g/mL natural products monomer and Nano medication；
（6-3）It is incubated after 24 h, discards culture medium, after being rinsed 2 ~ 3 times with pH 7.4 PBS solution, methanol is fixed
10min；
（6-4）Methanol is discarded, after drying, adds DAPI working solutions to wash the min of dyeing 5 ~ 10；
（6-5）Dyeing liquor is discarded, background 2 ~ 3 times is rinsed with PBS solution, then with distilled water flushing, is observed after drying, fluorescence microscopy
Mirror is observed, taken pictures with 340/380 nm ultraviolet excitations.
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