CN104623660B - Application of copper-doped gold nanorods in preparation of antitumor drugs - Google Patents
Application of copper-doped gold nanorods in preparation of antitumor drugs Download PDFInfo
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Abstract
The invention discloses an application of copper-doped gold nanorods in preparation of antitumor drugs. Studies of inventors show that copper-doped gold nanorods are converted into 2-5nm small-sized gold quantum dots under high-power ultrasound action and then selectively kill tumour cells, therefore, the copper-doped gold nanorods can be used as an online antitumor preparation for treating various cancers and the irradiation time of HIFU (high intensity focused ultrasound) can be shortened.
Description
Technical field
The present invention relates to a kind of new application of nano material, the more particularly to gold nanorods of copper doped are preparing antineoplastic agent
Answering in thing.
Background technology
Worldwide, cancer has become " first killer " of the mankind.World Health Organization (WHO)（WHO）Statistical data
（2012）Show, cancer causes 8,200,000 people dead.In following 20 years, estimate annual cases of cancer by by 1400 in 2012
Ten thousand rise to 22,000,000.The annual newfound cancer patient of China brings huge bearing more than 1,000,000 to patient and society
Load.
At present, treatment of the modern medicine to cancer is mainly operative treatment and coordinates Radiotherapy chemotherapy, part simultaneously using targeting or
Genomic medicine, is equipped with response Chinese traditional treatment and improves curative effect；For local or early-stage cancer, considerable part is poly- using laser or ultrasound
Burnt or alternation magnetic resonance thermotherapy treatment partial malignancy, or local operation.Though local operation can remove most of protopathy
Stove, but can not fundamentally prevent the regeneration and breeding of cancerous cell;Though Radiotherapy chemotherapy can kill cancerous cell, while also making a large amount of
Suffer damage including normal immunological histiocyte, induce bad gastrointestinal reaction, bone marrow depression and Liver and kidney, impairment of cardiac function, make
The body of patient is weaker, and resist the disease is greatly lowered with anti-infection ability, and majority is suffered from carninomatosis people and is difficult to successfully control
Treat or extend life cycle.
There are some researches show, the small size nano gold spherical of diameter 5 or 20nm is natural with good active anticancer
（Rochelle R. Arvizo et al, Inhibition of tumor growth and metastasis by a
Self-therapeutic nanoparticle, PNAS, 2013,110 (17): 176700–6705）, it is capable of achieving contracting within 3 days
It is little by 70~80%.But cancerous cell is low to the uptake ratio of gold nanosphere, cause its fragmentation effect not fully up to expectations.
Gold nanorods（Gold Nanorod, GNR）Itself is nontoxic, the characteristic with near-infrared laser response, can be used to swash
Photo-thermal therapy cancer, its weak point be gold nanorods easily because heat realize baseball change, cause its with near-infrared laser respond
Characteristic dies down and even disappear.And gold nanorods are changed into the oversized of gold nanosphere, also there is no anti-tumor activity.
The gold nanorods of copper doped are in the preparation process of gold nanorods, to be prepared by adding monovalence or bivalent cupric ion
A kind of new gold nanorods for obtaining, not there are some researches show that it has anti-tumor activity.
The content of the invention
It is an object of the invention to provide application of the gold nanorods of copper doped in antitumor drug is prepared.
Further object is that providing the anti-tumor agent that a kind of ultrasonic wave added treats tumor.
It is yet a further object of the present invention to provide a kind of preparation technology of gold nanosphere.
Inventor's research finds that the gold nanorods of copper doped can be converted into 2~5nm under the ultrasonication of higher-wattage
Small size gold quantum dot, so selective killing tumor cell and to normal cell basic nonhazardouss effect, improve patient's immunity
Ability.Experimental data shows that the gold nanorods of copper doped add the culture 48 of system containing hepatoma carcinoma cell little after supersound process
When, it acts on hepatoma carcinoma cell causes hepatoma carcinoma cell death more than 70%.The PEG of the gold nanorods of copper doped（Polyethylene
Glycol, Polyethylene Glycol）Dispersion liquid can be cultivated directly together with cancerous cell, in high intensity focused ultrasound（More than 1000W/cm2）
Under irradiation, that is, the strong golden quantum dot of 2~5nm small sizes, active anticancer is converted into, focuses on ultrasonic pyrogenicity and act on golden quantum dot
Under the dual function of cancerous cell, accelerate cancer cell death.Therefore this copper-golden nanometer rod of mixing can be used for as online anticancer preparation
Various cancers are treated, shortens HIFU（High Intensity Focused Ultrasound high intensity focused ultrasounds）During irradiation
Between.
Description of the drawings
Fig. 1 is the UV-Vis abosrption spectrograms for mixing copper GNR（a）, TEM figure（b）；
Fig. 2 is the scattered TEM figures for mixing copper GNR gained dispersion liquids Jing after 300W ordinary ultrasonics instrument acts on 20min of PEG；
Fig. 3 is the scattered TEM figures for mixing copper GNR Jing after different high intensity focused ultrasound devices are processed of PEG；
Fig. 4 is different draw ratios（Aspect Ratio, AR）The UV-Vis delustring spectrograms of PEG-GNR dispersion liquids；
Fig. 5 is impact figure of the different draw ratio PEG-GNR dispersion liquids incubations to hepatocellular carcinoma H22 cytoactive；
Fig. 6 be PEG- mix copper GNR dispersion liquids incubate Jing after different capacity supersound process to hepatocellular carcinoma H22 cell live
The impact figure of property；
Fig. 7 is different disposal to normal cell L02 and the impact figure of hepatocellular carcinoma H22 activity.
Specific embodiment
Conveniently to see, in the present invention, the gold nanorods for mixing copper are abbreviated as mixing copper GNR.
The gold nanorods of copper doped are preparing the application of antitumor drug.
In the gold nanorods of copper doped, the quality doping of copper is 0.5%~0.01%, further, the quality doping of copper
Measure as 0.2%~0.05%.
For the ease of target tumor, being coupled on the gold nanorods of copper doped has cancer target agent.Cancer target agent is preferably
Cancer target polypeptide.
In order to be beneficial to cellular uptake GNR, draw ratio AR of the gold nanorods of copper doped is more than 1.5.In view of preparation work
The complexity of skill, AR values are preferably 3~8.
The copper GNR that mixes of minor diameter is easier to be converted into gold nanosphere under ultrasonication, test result indicate that, diameter exists
20nm or following mix copper GNR in the presence of ultrasound can with higher rate conversion as gold nanosphere, but minor diameter
GNR have higher conversion ratio, consider, the diameter of gold nanorods is preferably 5~8nm.
A kind of anti-tumor agent, the gold nanorods containing copper doped.
In the gold nanorods of copper doped, the quality doping of copper is 0.5%~0.01%, further, the quality doping of copper
Measure as 0.2%~0.05%.
A kind of preparation technology of golden quantum dot, including：The gold nanorods of copper doped are dispersed in dispersant, are carried out afterwards
Supersound process, obtains golden quantum dot.
In the gold nanorods of copper doped, the quality doping of copper is 0.5%~0.01%, further, the quality doping of copper
Measure as 0.2%~0.05%.
Mix the preparation of copper GNR
Mixing copper GNR can be using crystal seed method, such as document Xingchen Ye et al, Improved Size-Tunable
Synthesis of Monodisperse Gold Nanorods through the Use of Aromatic
Additives, ACS nano 2012,6 (3):Method disclosed in 2804-2817 is prepared；Non- crystal seed can also be adopted
Method, the method as disclosed in inventor is in CN 103203459A is prepared.
For the sake of convenient, the example that copper GNR is mixed in present invention preparation is as follows with method：
5mL 0.1M CTAB are taken in 25mL beakers, 500 μ L chlorauric acid solutions are added toward beaker, added again in stirring
Enter 32.5 μ L silver nitrate solutions, 50 μ L ascorbic acid solutions, stir, add 3 μ L sodium borohydride solutions, proceed to 30 DEG C of water
Water-bath 11min in bath, adds 5mL deionized waters, 32.5 μ L silver nitrate solutions in stirring, add 5 μ L 0.1M CuCl
Dispersion liquid, proceeds to 22 DEG C of cooling circulating slot growth 24h.It is distributed to that to obtain final product PEG in PEG solution scattered after centrifugation, cleaning
Mix copper GNR solution.
What the method was prepared mixes UV-Vis absorption spectrums such as Fig. 1 of copper GNR（a）It is shown, its TEM figure such as Fig. 1（b）
It is shown.It is 0.1% that fluorimetry measures wherein copper content.By the consumption for changing CuCl dispersion liquids or other cupric predecessors
Or concentration is capable of achieving to mix amount of copper in GNR.
Mix the supersound process characteristic of copper GNR：
Take 500 μ L optical density (OD)s（OD）Copper GNR is mixed for 4（LSPR=803nm, copper content 0.1%, similarly hereinafter）PEG disperses
Liquid, in being put into common 300W, 50Hz Ultrasound Instrument, ultrasonic 20min.
The TEM figures for mixing copper GNR before and after sonicated are respectively such as Fig. 1（b）, shown in Fig. 2, Fig. 2（a）In, the upper left corner
Illustration is the TEM pictures after the supersound process amplified, and remainder is the TEM figures after processing.From Fig. 2（a）In it can also be seen that
After mixing the supersound process of copper GNR Jing higher-wattages, 2~5nm gold quantum dots are converted into.And to undoped p under the same terms
GNR carries out supersound process, and the pattern of gold nanorods does not occur visible change.
It is further test result indicate that, when the quality doping of copper is 0.5%~0.01%, mixing copper GNR can be super
Golden quantum dot is converted in the presence of sound.When the quality doping of copper is 0.2%~0.05%, when more preferably 0.1%, copper GNR is mixed
Preferably sonicated can be converted into golden quantum dot.Fig. 2（b）Give that to mix the dispersion liquid Jing that copper mass is 0.01% common
The TEM figures of 300W, 50Hz Ultrasound Instrument ultrasound 20min.
Mix the HIFU process of copper GNR
Take 500 μ L optical density (OD)s（OD）For the GNR for mixing copper 0.1% of 4 PEG modifications（LSPR=800nm）Dispersion liquid, puts
In entering 5mL PP pipes, the JC type focusing ultrasound tumor treating systems that Chong Qinghai helps company to produce are inserted, select frequency 1.4MHz, it is burnt
Domain meansigma methodss：1.2mm；Focal length 135mm；The W of power 220, action time 2 min, gained dispersion liquid is golden quantum dot.Equally, adopt
The type high-intensity focus supersonic tumor treatment systems of HIFU 1 produced with Xin Di industrial corporations of Shanghai Communications University, focus power control
System is in 1200W/cm2, irradiation 1min, gained dispersion liquid also switchs to golden quantum dot.Fig. 3 sets forth its TEM figure（A, b）.Can
To find out, due to focussing force, the golden quantum dot that copper GNR is converted into quickly at short notice irregular, a diameter of 2~5nm is mixed.
Mix the antitumor action of copper GNR
The application for mixing copper GNR in anticancer aspect of the present invention is expanded on further below by way of experiment.
The PEG dispersion liquids of GNR are abbreviated as PEG-GNR.
Mtt assay main process in the following example is as follows：
1) logarithmic (log) phase cell is collected, adjusts concentration of cell suspension, add 100 ml, bed board to make cell to be measured adjust density per hole
To 5000/ hole,（Edge hole is filled with aseptic PBS）；
2)5%CO2, 37 DEG C are incubated, and to cell monolayer bottom hole is paved with（96 hole flat undersides）, 12h is cultivated, after cell attachment,
When growth conditions are good, remove old culture fluid, take out with containing/or without, Jing/or not sonicated setting concentration
Mix copper/or do not mix copper-golden nanometer rod（Pollution is prevented before PEG-GNR uses with ultra-vioket radiation sterilization 30min）Culture fluid
The cell of culture certain hour, per the μ l of hole 100, if 5 multiple holes；
3) continue in 5%CO2, certain hour is incubated under the conditions of 37 DEG C, observe under inverted microscope；
4) culture plate is taken out, 20 μ l MTT solution (5mg/ml, i.e. 0.5%MTT) is added per hole, continue to cultivate 4h；
5) terminate culture, carefully suck culture fluid in hole；
6) 150 μ l dimethyl sulfoxide are added per hole, the min of low-speed oscillation 10 on shaking table is put, crystal is fully dissolved,
The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument OD 490nm；
7) while it is blank group to arrange zeroing hole（Culture fluid, MTT, dimethyl sulfoxide）, control wells（Cell, same concentrations
GNRs dissolving medium PEG, culture fluid, MTT, dimethyl sulfoxide）；
8) interpretation of result:Survivaling cell %=（OD experimental group-OD blank groups）/（OD compares-OD blank groups）×100%.
If not specializing below, copper GNR copper contents of mixing used represent gold nano grain for 0.1%, GNP（Gold
Nanoparticle）.
The cytotoxicity of undoped p GNR dispersion liquid
Cell is the hepatoma Hep G 2 cells for cultivating, and period adds Jing 300W Ultrasound Instrument ultrasound 30min, different draw ratios
(its UV-Vis Spectral Extinction is as shown in Figure 4 for PEG-GNR dispersion liquids）So as to middle concentration be 12.5,25,37.5,50nM, observation temperature
The activity of hepatocellular carcinoma H22 after 24h, 36h is educated, as a result such as Fig. 5（A, b）.As a result show, different draw ratios it is unadulterated
GNR cytotoxicities are more or less the same, consistent with the result that document discloses report to the basic avirulence of hepatoma Hep G 2 cells.
PEG- mixes the cytotoxicity of copper GNR dispersion liquids after supersound process
Using mtt assay, first the copper PEG-GNR different capacity ordinary ultrasonics instrument of mixing that draw ratio is 4.7 is disperseed into 30min,
The relation of re-test hepatoma Hep G 2 cells activity vs. concentration and incubative time.As a result such as Fig. 6.Fig. 6 results show, with 100
To the almost non-toxic difference of cancerous cell after W is ultrasonic, after 300 W ultrasound 30min, mix copper GNR dispersion liquids has aobvious to hepatoma Hep G 2 cells
Write toxicity；And, this toxicity mixes the concentration of copper GNR dispersion liquids, cultivation time with addition PEG- to be increased and enhancing, 48h（2d）
Afterwards, hepatoma Hep G 2 cells activity is reduced to 10%.Thus illustrate, it is high-power that PEG- mixes copper GNR dispersion liquid Jing（300W）It is common super
After sound, active anticancer strengthens, and has persistence to hepatoma Hep G 2 cells toxicity, and it is special that hint mixes copper GNR Jing after large power supersonic
Property is significantly changed；And low-power（100 usual W of laboratory）Ultrasound Instrument ultrasound same time, almost without cell
Toxicity, implies that the pattern for now mixing copper GNR has no and significantly changes.
Copper GNR is mixed under high-power ultrasonic effect to normal cell L02 and hepatoma Hep G 2 cells using mtt assay research
Toxicity, addition mixed in the test group of copper GNR, and the concentration for mixing copper GNR is 250 nM, as follows under packet and disposition：
First group of cell：Matched group, without the culture fluid culture 72 hours for mixing copper GNR and not through supersound process；
Second group of cell：Merely through the ultrasonic Treatment 15 minutes of 300W；
3rd group of cell：The culture fluid culture 72 hours of copper GNR is mixed only with addition, not through supersound process；
4th group of cell：After the culture fluid culture 72 hours of copper GNR is first mixed using addition, the culture for mixing copper GNR will be contained
Liquid is removed, and is substituted with the culture fluid without GNR, then through 300W ultrasonic Treatment 15 minutes；
5th group of cell：After the culture fluid culture 72 hours of copper GNR is first mixed using addition, culture fluid is directly super using 300W
Sonicated 15 minutes.
Experimental result is as shown in Figure 7.It is specific as follows：
In second group of cell, the survival rate of two kinds of cells drops to respectively 86.9% and 84.5%, shows high-power ultrasonic
Cell is deposited with certain damage, but affect difference unobvious；
In 3rd group of cell, the survival rate of two kinds of cells is respectively 74% and 75%, and the two difference is not also obvious.Because culture
The concentration that copper GNR is mixed in liquid has reached 250 nM, therefore has certain toxicity to cell；
In 4th group of cell, the survival rate of two kinds of cells is down to respectively 44.3% and 57%.
In 5th group of cell, the survival rate of two kinds of cells is down to respectively 21% and 41%.
Interpretation of result：
In fact, high-power ultrasonic and high concentration are mixed, impacts of the copper GNR to hepatoma carcinoma cell survival rate is estimated should to be
86.9%´74%=64.3%.However, carrying out ultrasonic Treatment again after we allow cellular uptake to mix copper GNR, the survival of cell is found
Rate only has 44.3%（Remove containing the culture fluid for mixing copper GNR before ultrasonic Treatment）With 21%（Retain to contain before ultrasonic Treatment and mix copper GNR
Culture fluid）, illustrate to mix small size GNP that copper GNR is transformed under high-power ultrasonic effect cell is generated it is certain
Toxicity.Because the intake of cell is limited, therefore the former survival rate of cell is greater than the latter.
For normal liver cell, it is pre- that high-power ultrasonic and high concentration mix impacts of the copper GNR to hepatoma carcinoma cell survival rate
Meter should be 84.5% 75%=63.4%.However, carrying out ultrasonic Treatment again after we allow cellular uptake to mix copper GNR, find thin
The survival rate of born of the same parents is 57.1%（Remove containing the culture fluid for mixing copper GNR before ultrasonic Treatment）With 41%（Retain before ultrasonic Treatment and contain
Mix the culture fluid of copper GNR）, also explanation mixes copper GNR small sizes GNP for being transformed under high-power ultrasonic effect and cell produced
Certain toxicity.
As can be seen that in the case where the collective effect of copper GNR and high-power ultrasonic is mixed, the survival rate of normal liver cell will
More than the survival rate of hepatoma carcinoma cell, particularly when without the culture fluid containing GNR is removed, illustrate that normal liver cell has certain
Self-repair function.Research before shows that GNR can be excluded body by normal cell after intake GNR through a period of time
Outward.Therefore, the GNR negligible amounts in normal cell are remained in so that under ul-trasonic irradiation produce GNP quantity also compared with
Little, the impact to cell survival rate is also just substantially little than hepatoma carcinoma cell.
Claims (6)
1. application of the gold nanorods of copper doped in antitumor drug is prepared, wherein, in the gold nanorods of copper doped, the matter of copper
Amount doping is 0.01%~0.5%, and the draw ratio of gold nanorods is more than 1.5, a diameter of 5~8nm of gold nanorods.
2. application according to claim 1, it is characterised in that：In the gold nanorods of copper doped, the quality doping of copper is
0.05%~0.2%.
3. application according to claim 1 and 2, it is characterised in that：Being coupled on gold nanorods has cancer target agent.
4. a kind of preparation technology of golden quantum dot, including：The gold nanorods of copper doped are dispersed in dispersant, are surpassed afterwards
Sonication, obtains golden quantum dot；Wherein, in the gold nanorods of copper doped, the quality doping of copper is 0.01%~0.5%, Jenner
The draw ratio of rice rod is more than 1.5, a diameter of 5~8nm of gold nanorods.
5. preparation technology according to claim 4, it is characterised in that：In the gold nanorods of copper doped, the quality doping of copper
Measure as 0.2%~0.05%.
6. a kind of anti-tumor agent, it is characterised in that：Gold nanorods containing copper doped in the preparation；Wherein, copper doped
In gold nanorods, the quality doping of copper is 0.01%~0.5%, and the draw ratio of gold nanorods is more than 1.5, gold nanorods it is straight
Footpath is 5~8nm.
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| A Seed-Based Diffusion Route to Monodisperse Intermetallic CuAu Nanocrystals;Wei Chen,et al;《Nanoparticles》;20100315;第49卷(第16期);第2917-2921页 * |
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