CN103933583B - Preparation of chiral gold nanocluster of suppression MGC-803 cell and application thereof - Google Patents
Preparation of chiral gold nanocluster of suppression MGC-803 cell and application thereof Download PDFInfo
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- CN103933583B CN103933583B CN201410147843.4A CN201410147843A CN103933583B CN 103933583 B CN103933583 B CN 103933583B CN 201410147843 A CN201410147843 A CN 201410147843A CN 103933583 B CN103933583 B CN 103933583B
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Abstract
The invention discloses a kind of preparation of chiral gold nanocluster suppressing MGC-803 cell and application thereof；Gold chloride and D-GSH that mol ratio is 1:3 are placed in ultra-pure water, through mixed at room temperature and stirring, are placed in ice bath and are cooled to 0 DEG C, form white suspension；Rapidly join tetrabutyl ammonium borohydride solution, and ice bath is stirred vigorously, form the solution of the gold nano cluster wrapped up containing D-GSH；Centrifugal segregation insoluble matter, the gold nano cluster in supernatant is precipitated by adding the cold ethanol of excess, centrifugal collection gold nano cluster, repeats methanol precipitation step several times, lyophilization,.The gold nano cluster of present invention synthesis has good water solublity and stability, and maximum emission peak is in 650 nanometers, and maximum excitation peak is in 520 nanometers.Can effectively stop the cell cycle of MGC-803, wherein G2The cell proportion of/M phase is significantly increased, and finally promotes the necrosis of MGC-803 cell.
Description
Technical field
The present invention relates to preparation and the biomedical applications of technical field of pharmaceuticals, be specifically related to a kind of suppression MGC-803 cell
The preparation of chiral gold nanocluster and application thereof.
Background technology
Gastric cancer is one of common malignant tumor of China, occupies the second of all kinds of tumor, mortality rate pole at its sickness rate of China
High.How asymptomatic early gastric cancer is or only light symptoms, and when clinical symptoms is obvious, pathological changes is many has belonged to late period.Therefore,
Being diagnosed to be the early symptom of gastric cancer in time, seek medical advice in time, the diagnosis and treatment to gastric cancer have very important significance.Receive in recent years
Rice technology and applications to nanostructures be targets identification and treatment in-vivo tumour provide good method.Tumor at present
The subject matter of clinical diagnosis and treatment is: the Clinics and Practices of tumor separates, and tumor can not effectively be distinguished by overwhelming majority cancer therapy drug
Cell and normal cell, result brings systematic toxicity and other side effect.Although nano target medicine delivers now
Systematic research develops quickly, but generally there is the shortcomings such as probe builds complexity, and repeatability is unstable.
In recent years, the controlled preparation of gold nano cluster and biomedical applications are more and more paid attention to, Rongchao
Jin etc. are controlled by kinetics and thermodynamic (al) condition, controllable precise synthesis Au25Gold nanoclusters (Wu, Z., MacDonald,
M.A.,Chen,J.,Zhang,P.,&Jin,R.(2011).Journal of the American Chemical
Society,133(25),9670-9673)；In addition Jianping Xie et al. utilizes bovine serum albumin BSA conduct
Template, the alkaline aqueous solution of 37 DEG C has synthesized high quantum production rate gold nano cluster (Xie, J., Zheng, Y. ， &Ying,
J.Y.(2009).Journal of the American Chemical Society,131(3),888-889)。Li
The survey article that Shang delivers on Nano Today points out that gold nano cluster is owing to having extra small particle diameter, well
The advantages such as the light stability of biocompatibility and excellence, ultrasensitive biological Molecular Detection, living things system fluorescent labeling with
And other biological medical domain has great potential using value.Find in existing technology through retrieval, despite very
Utilize biomolecule such as protein and nucleic acid a lot of to the document synthesizing fluorogold nanocluster as protective agent more, but at present
The most do not use the glutathione molecules of nature uncommon D configuration to synthesize the fluorescence emission peak Jenner in 650 nanometers
The report of rice cluster.Find in existing technology through retrieval, use despite report the glutathione molecules of D configuration to close
Become CdTe (cadmium telluride) quantum dot, but due to the heavy metal toxicity of Cd element, seriously limit it in living things system
Application, and we synthesis gold nano cluster do not introduce any poisonous element and material, wherein gold element has very
Good biocompatibility.The gold nanoclusters of the glutathione molecules synthesis of D configuration is than the L structure generally existed in living things system
The gold nano cluster of the glutathione molecules synthesis of type more effectively suppresses growth and the propagation of stomach cancer cell MGC-803, and
Remarkably promote the death of MGC-803 cell, this inhibitory action in normal gastric epithelial cell GES-1 the most almost
No, therefore, the gold nanoclusters of the glutathione molecules synthesis of the D configuration of preparation has specific suppression gastric cancer tumor
The effect of cell MGC-803, is the nano material of a kind for the treatment of gastric cancer having application prospect.
Summary of the invention
Present invention aims to the deficiency that above-mentioned prior art exists, it is provided that a kind of chiral gold suppressing MGC-803
Preparation of nanocluster and application thereof.It is specifically related to a kind of chiral gold nanoclusters wrapped up with the glutathione molecules of D configuration
Bunch synthesis, and as the specific purposes carrying out stomach cancer cell MGC-803 killing treatment of nano-probe.
For achieving the above object, the present invention realizes by the following technical solutions:
First aspect, the present invention relates to the preparation method of a kind of chiral gold nanocluster suppressing MGC-803, described method
Comprise the steps:
A, gold chloride and D-GSH that mol ratio is 1:3 are placed in ultra-pure water, through mixed at room temperature and stirring, are placed in ice
Bath is cooled to 0 DEG C, forms white suspension；
B, rapidly joining tetrabutyl ammonium borohydride solution in described white suspension, and ice bath is stirred vigorously, and formation contains
There is the solution of the gold nano cluster that D-GSH wraps up；
Insoluble matter in C, centrifugal segregation step B gained solution, the gold nano cluster in supernatant is by adding excess
Cold ethanol is precipitated, and is then centrifuged for collecting gold nano cluster, repeats methanol precipitation step several times, lyophilization,
Obtain described chiral gold nanocluster solid.
Preferably, in step A, the concentration of described chlorauric acid solution be the concentration of 10mM, D-GSH be 150mM；Institute
The time stating mixing and stirring is 20 minutes.
Preferably, in step B, described tetrabutyl ammonium borohydride is 5:1 with the mol ratio of gold chloride.
Preferably, in step B, being stirred vigorously speed is 1200～2000rpm, and mixing time is 1～4 hour.
Preferably, in step C, described precipitation is particularly as follows: with 3 times of volumes in the cold ethanol of gold nano cluster supernatant
Precipitate, and 10000rpm is centrifuged 15 minutes.
Second aspect, the invention still further relates to the prepared chiral gold nanocluster of a kind of above-mentioned method and is suppressing as specificity
Stomach cancer cell MGC-803 growth and the purposes in the nano-probe of propagation.
The third aspect, the invention still further relates to the prepared chiral gold nanocluster of a kind of above-mentioned method as promoting that gastric cancer is thin
Purposes in the nano-probe that born of the same parents MGC-803 is dead.
Fourth aspect, the invention still further relates to the prepared chiral gold nanocluster of a kind of above-mentioned method in preparation treatment gastric cancer
Purposes in nano target medicine.
It is a discovery of the invention that the gold nanoclusters of the glutathione molecules synthesis of D configuration is than the L structure generally existed in living things system
The gold nano cluster of the glutathione molecules synthesis of type more effectively suppresses growth and the propagation of stomach cancer cell MGC-803, this
Kind of inhibitory action in normal gastric epithelial cell GES-1 but almost without, therefore, the paddy Guang of the D configuration of preparation
The gold nanoclusters of sweet peptide molecule synthesis has the effect of specific suppression gastric cancer tumor cell MGC-803, is that one has should
Nano material by the treatment gastric cancer of prospect.
Compared with prior art, there is advantages that the present invention is simple to operate, reaction condition is gentle, closes
The gold nano cluster become has good water solublity and stability, and maximum emission peak is in 650 nanometers, and maximum excitation peak is 520
Nanometer.Can effectively stop the cell cycle of MGC-803, wherein G2The cell proportion of/M phase is significantly increased, and finally
Promote the necrosis of MGC-803 cell.
Accompanying drawing explanation
By the detailed description non-limiting example made with reference to the following drawings of reading, the further feature of the present invention,
Purpose and advantage will become more apparent upon:
Fig. 1 is the high resolution TEM figure of the gold nano cluster of the glutathione synthesis of the D configuration in embodiment 1；
Fig. 2 is the gold nano cluster circular dichroism spectrogram of the glutathione synthesis of L Yu the D configuration in embodiment 2；
Fig. 3 is that the gold nano cluster of the glutathione synthesis of the D configuration in embodiment 3 is to MGC-803 cell cycle
Retardance figure；Wherein, upper figure is not add the matched group that gold nano cluster processes, and figure below is the D configuration of 500 μ g/ml
The retardance to MGC-803 cell cycle of the gold nano cluster of glutathione synthesis；
Fig. 4 be in embodiment 4 gold nano cluster of the glutathione synthesis of D configuration to MGC-803 Yu GES-1 cell
The impact of survival rate；
Fig. 5 is the detection of MGC-803 Yu GES-1 cell GSTP1 promoter region methylation state.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.Following example will assist in those skilled in the art
Member is further appreciated by the present invention, but limits the present invention the most in any form.It should be pointed out that, the common skill to this area
For art personnel, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement.These broadly fall into
Protection scope of the present invention.
The present invention passes through addition and the low temperature environment of ice bath of weak reductant tetrabutyl ammonium borohydride, reduces building-up process
In nucleation and the speed of growth, make the gold nano cluster uniform particle diameter of synthesis have good dispersibility, and excellent
Fluorescent optics character.Part in building-up process is respectively adopted chiral glutathion L-GSH and D-GSH, wherein
L-GSH and D-GSH enantiomer each other, the structure of L-GSH consists of
N-γ-L-glutamyl-L-cysteinyl-glycine, the structure of D-GSH consists of
N-γ-D-glutamyl-D-cysteinyl-glycine.By two kinds of good to synthesis purification chiral gold nanoclusters respectively
Hatch altogether with people gastric epithelial cell GES-1 with gastric carcinoma cells MGC-803, wherein the gold nano group of D-GSH synthesis
Bunch MGC-803 cell is had the growth inhibited effect of concentration dependant, and the significantly growth of GES-1 cell is pressed down
Make and use.Further investigations have shown that the high methylation of MGC-803 cell GSTP1 promoter region makes GSTP1
Expression be suppressed, it is impossible to play Scavenger of ROS function, and D-GSH synthesis gold nano cluster (AuNCs@D-GSH)
The gold nano cluster (AuNCs@L-GSH) comparing L-GSH synthesis after entering gastric cancer cell line MGC-803 can produce obvious oxygen
Changing stress, the generation of a large amount of active oxygens and the defect of the expression of GSTP1, in making the mitochondrion of MGC-803 cell
Transmembrane potential reduces, and cell cycle is obstructed, then downright bad.It is specifically shown in following embodiment:
Embodiment 1
(1) gold chloride and the preparation of D type glutathione molecules mixed liquor
It is incorporated in room temperature 25 DEG C by mixed with the D type glutathione solution of 2ml 150mM for the chlorauric acid solution of 10ml 10mM
Lower stirring 20 minutes.It is subsequently placed in ice bath and is cooled to 0 DEG C, form the suspension of white.
(2) in ice bath environment, gold nano cluster is synthesized by the reduction of weak reductant tetrabutyl ammonium borohydride
In the suspension of above-mentioned white, rapidly join tetrabutyl ammonium borohydride solution, and ice bath be stirred vigorously 4 hours after
Gold nano cluster to D-GSH parcel.
The amount of the tetrabutyl ammonium borohydride solution added is 166.67mM, 3ml, and synthesis temperature condition is ice bath, stirring speed
Degree is 1200-2000rpm, and continuing mixing time is 4 hours
(3) the 3rd steps, are led to the insoluble matter of solution after synthesis by centrifugal segregation, the gold nano cluster in supernatant solution
The cold ethanol crossing addition excess is precipitated, and is then centrifuged for collecting gold nano cluster, repeats methanol precipitation step twice,
Obtain pure gold nano cluster, and lyophilization obtains gold nano cluster solid.(the gold nano of L-type glutathion parcel
Cluster is synthesized by above-mentioned method.)
Referring to of the pelleting centrifugation gold nano cluster told: sink in the cold ethanol of gold nano cluster supernatant with 3 times of volumes
Form sediment, and 10000rpm is centrifuged 15 minutes.Repeat above methanol precipitation step twice, obtain gold nano cluster after purification.
By the gold nano cluster ultrathin carbon films copper mesh sample preparation of above-mentioned preparation, JEM-2100F Flied emission high-resolution-ration transmission electric-lens
Observing gold nano cluster particle size and pattern, transmission electron microscope picture is shown in Fig. 1.As shown in Figure 1, gold nano cluster particle diameter is equal
One and diameter about 2 nanometer.
Embodiment 2, the mensuration of gold nano cluster chirality
The circular dichroism spectra of the gold nano cluster of synthesis measures on Jasco J-815 type circular dichroism spectrometer, and temperature is room temperature
25 DEG C, a length of 10mm of light path of quartz colorimetric utensil used, carry a width of 1nm, scanning speed is 50nm/min, scans model
Enclosing for 200-500nm, the homogenizing value taking scanning three times is end value, and spectrogram is shown in Fig. 2.As shown in Figure 2, D Yu L structure
The circular dichroism spectra signal of the gold nano cluster of the glutathione synthesis of type has the feature of perfect specular, illustrates respectively
Successfully synthesize the gold nano cluster of the glutathione molecules parcel of two kinds of configurations.
Embodiment 3, the mensuration of cell cycle
After gold nano cluster synthetic for embodiment 1 and MGC-803 cell are hatched 12 hours altogether, measure cell cycle.
Described the cell cycle method is: by MGC-803 cell according to 2 × 104The density in/hole is planted in six orifice plates,
Adding a certain amount of gold nano cluster after adherent, making gold nano cluster solution ultimate density is 500 μ g/ml, hatches 12 altogether
After hour, trypsinization centrifugal collecting cell, the cell that every hole is collected adds the ethanol of the 70% of 1ml pre-cooling, gently
Piping and druming mixing, fixes 24 hours for 4 DEG C.About 1000g collects cell in centrifugal 3-5 minute, and adds the PBS of 1ml pre-cooling
Re-suspended cell.Often pipe addition iodate third is stung the concentrated solution of (PI) and RNaseA and fully mixes, and makes PI final concentration of 50
The final concentration of 100 μ g/ml of μ g/ml, RNaseA, 37 DEG C of lucifuges hatch flow cytomery after 30 minutes.Streaming
Result is shown in Fig. 3.The matched group from the figure 3, it may be seen that compare, the gold nano of the glutathione synthesis of 500 μ g/ml D configurations
The MGC-803 cell that cluster processed is in the ratio of G2/M phase and is significantly increased.
Embodiment 4, the mensuration of cell survival rate
Gold nano cluster synthetic for embodiment 1 is hatched respectively altogether 24 hours, so with MGC-803 and GES-1 cell
Afterwards with MTT colorimetric method for determining cell survival rate.
Described incubation method altogether is: by MGC-803 Yu GES-1 cell according to 5 × 103It is thin that the density in/hole is planted in 96 holes
In born of the same parents' culture plate, adherent after add a certain amount of gold nano cluster, the ultimate density of gold nano cluster solution is 10,50,
100,250,500,1000 μ g/ml, each concentration arranges three multiple holes, and matched group is not add gold nano cluster
The normal cell cultivated.After hatching 24 hours altogether, MTT colorimetric method for determining cell survival rate.Cell survival rate is shown in Fig. 4.
As shown in Figure 4, under identical concentration, the MGC-803 that the gold nano cluster of the glutathione synthesis of D configuration processed
The cell that the survival rate of cell processed significantly lower than the gold nano cluster of the glutathione synthesis of L-configuration.
Embodiment 5, MGC-803 Yu GES-1 cell GSTP1 promoter region methylation state detect
The genomic DNA extracting MGC-803, GES-1 and MCF-7 cell respectively carries out GSTP1 promoter region methyl
Changing state-detection, wherein the MCF-7 cell of GSTP1 promoter region high methylation position is as positive control, with water is
Sample is as negative control.Above-mentioned sample uses EZ DNA methylation test kit-Gold, and (buying is opened from Beijing Tian Mo science and technology
Send out company limited) process, take 500ng genomic DNA to carrying out step in PCR pipe and in PCR instrument
Operation: (1) 98 DEG C of mode 10 minutes；Place 2 hours for (2) 64 DEG C；(3) 4 DEG C it are placed at once two hours.At Jing Guo
The DNA sample that reason changes is purified according to the step in test kit description, DNA sample after purification of then learning from else's experience
1/10 carry out methylation status of PTEN promoter as template.The operating procedure of methylation status of PTEN promoter is: 95 DEG C of total degeneration
3 minutes, then 94 DEG C of degeneration 30s, 58 DEG C of annealing 30s, 72 DEG C extended 30s, and carry out 35 circulations, last 72 DEG C
Extend 10 minutes.Wherein the forward sequence of GSTP1 site methylated primers is: 5'-TTCGGGGTGTAGCGGTCGTC-3'
(as shown in SEQ ID NO.1), reverse sequence is: 5'-GCCCCAATACTAAATCACGACG-3'(such as SEQ ID NO.2
Shown in)；The forward sequence of the non-methylated primers in GSTP1 site is: 5'-GATGTTTGGGGTGTAGTGGTTGTT-3'(is such as
Shown in SEQ ID NO.3), reverse sequence is: 5'-CCACCCCAATACTAAATCACAACA-3'(such as SEQ ID NO.4
Shown in).PCR primer separates in 12% polyacrylamide gel electrophoresis, observes and take pictures after ethidium bromide staining under uviol lamp,
See Fig. 5；As shown in Figure 5, the GSTP1 promoter region high methylation of MGC-803 cell.
Above the specific embodiment of the present invention is described.It is to be appreciated that the invention is not limited in
Stating particular implementation, those skilled in the art can make various deformation or amendment within the scope of the claims,
This has no effect on the flesh and blood of the present invention.
Claims (6)
1. the preparation method of the chiral gold nanocluster suppressing MGC-803 cell, it is characterised in that described method
Comprise the steps:
A, gold chloride and D-GSH that mol ratio is 1:3 are placed in ultra-pure water, through mixed at room temperature and stirring, are placed in ice
Bath is cooled to 0 DEG C, forms white suspension；
B, rapidly joining tetrabutyl ammonium borohydride solution in described white suspension, and ice bath is stirred vigorously, and formation contains
There is the solution of the gold nano cluster that D-GSH wraps up；
Insoluble matter in C, centrifugal segregation step B gained solution, the gold nano cluster in supernatant is by adding excess
Cold ethanol is precipitated, and is then centrifuged for collecting gold nano cluster, repeats methanol precipitation step several times, lyophilization,
Obtain described chiral gold nanocluster solid.
The preparation method of the chiral gold nanocluster of suppression MGC-803 cell the most according to claim 1, it is special
Levy and be, in step A, the concentration of described chlorauric acid solution be the concentration of 10mM, D-GSH be 150mM；Described mixing
It it is 20 minutes with the time of stirring.
The preparation method of the chiral gold nanocluster of suppression MGC-803 cell the most according to claim 1, it is special
Levying and be, in step B, described tetrabutyl ammonium borohydride is 5:1 with the mol ratio of gold chloride.
The preparation method of the chiral gold nanocluster of suppression MGC-803 cell the most according to claim 1, it is special
Levying and be, in step B, being stirred vigorously speed is 1200～2000rpm, and mixing time is 1～4 hour.
The preparation method of the chiral gold nanocluster of suppression MGC-803 cell the most according to claim 1, it is special
Levy and be, in step C, described precipitation particularly as follows: with 3 times of volumes in the cold ethanol precipitation of gold nano cluster supernatant,
And 10000rpm is centrifuged 15 minutes.
6. the chiral gold nanocluster that the method for claim 1 prepares is at the nanometer target of preparation treatment gastric cancer
Purposes in medicine.
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| CN106075469A (en) * | 2016-06-13 | 2016-11-09 | 上海交通大学 | Gd3+induction gold nano cluster is self-assembled into method and the application of gold nano grain |
| DK3449945T3 (en) * | 2016-08-05 | 2022-03-07 | Shenzhen Profound View Pharma Tech Co Ltd | SUBSTANCE CONTAINING GOLD CLUSTER AND MANUFACTURING PROCEDURE AND USE |
| CN112190767B (en) * | 2020-09-22 | 2022-07-26 | 上海市第六人民医院 | Nano-antibacterial coating material based on nanogold cluster and preparation method thereof |
| CN113230400A (en) * | 2021-03-11 | 2021-08-10 | 上海交通大学 | Gold nanocluster radiotherapy sensitization assembly based on epigenetic regulation and control as well as preparation and application thereof |
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| US20050074551A1 (en) * | 2002-08-01 | 2005-04-07 | Xueying Huang | Ethylene glycol monolayer protected nanoparticles |
| CN101538736A (en) * | 2008-03-17 | 2009-09-23 | 国家纳米科学中心 | Dendritic golden nanophase material and preparation method thereof |
| CN101710076A (en) * | 2009-12-29 | 2010-05-19 | 东北师范大学 | Lead ion colorimetric detection probes and application method thereof |
| CN102614535A (en) * | 2011-02-01 | 2012-08-01 | 中原大学 | Medical contrast agent and manufacuture method for microbubbles containing fluorescent gold nanoclusters |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050074551A1 (en) * | 2002-08-01 | 2005-04-07 | Xueying Huang | Ethylene glycol monolayer protected nanoparticles |
| CN101538736A (en) * | 2008-03-17 | 2009-09-23 | 国家纳米科学中心 | Dendritic golden nanophase material and preparation method thereof |
| CN101710076A (en) * | 2009-12-29 | 2010-05-19 | 东北师范大学 | Lead ion colorimetric detection probes and application method thereof |
| CN102614535A (en) * | 2011-02-01 | 2012-08-01 | 中原大学 | Medical contrast agent and manufacuture method for microbubbles containing fluorescent gold nanoclusters |
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