CN103861124B - The preparation method of platinum cluster and the application in the preparation and apoptosis agent of tumor - Google Patents
The preparation method of platinum cluster and the application in the preparation and apoptosis agent of tumor Download PDFInfo
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- CN103861124B CN103861124B CN201410075034.7A CN201410075034A CN103861124B CN 103861124 B CN103861124 B CN 103861124B CN 201410075034 A CN201410075034 A CN 201410075034A CN 103861124 B CN103861124 B CN 103861124B
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Abstract
The invention discloses the preparation method of platinum cluster and the application in the preparation and apoptosis agent of tumor.Concrete steps: the saline solution of platinum and ascorbic acid (VC) and reductive glutathione (GSH) mixing heating in water bath, control to receive the aggregate velocity of platinum rice bunch and particle diameter by regulation pH value of solution and temperature and each component ratio.The platinum cluster material particle size that the present invention prepares is little, good penetrability, has excellent biocompatibility, stable fluorescent characteristic, it is possible to achieve to the high time-space resolution imaging of the lesions positions such as tumor and be effectively facilitated apoptosis of tumor cells and tumor tissues melts；The present invention method application simple and easy to do, toxicity is low, highly sensitive, multi-modal real-time dynamic monitoring can be carried out simultaneously, there is wide clinical medicine using value and prospect.
Description
Technical field
The present invention relates to the preparation method of a kind of platinum cluster and the application in the preparation and apoptosis agent of tumor.This
The relevant platinum cluster preparation that bright utilization prepares precisely guides the disease site such as tumor as probe, and can be further combined with by becoming
Light heating therapy as guidance, it is achieved highly sensitive diagnosis and the treatment to tumor.
Background technology
At present, the death of malignant tumor (cancer) still occupies first of all kinds of cause of the death in the world, becomes and captures people now
A big killer of life.Owing to cancer early symptom is inconspicuous, it is difficult to be found early, thus the early diagnosis of cancer and controlling
Treating and be still one of most important medical problem urgently to be resolved hurrily, the healing to cancer is the most crucial.
In recent years, along with the development of Nanometer scale science and technology, some special natures that nano material possesses because of himself
The research for the treatment of of cancer gets most of the attention.Nano material is little due to its particle diameter, easily by barrier cell, and due to tumor
The enhanced permeability and retention effect that the hyperfunction and unsound lymphatic drainage system of tissue microvascular permeability produces so that it is prone to excellent
First assembling at tumor locus, additionally metal nano material can raise with the raw free radical of absorbing light and delivery in hot weather or temperature, thus ten
Imaging and the light thermotherapy etc. that are conducive to tumor are divided to act on.But during light thermotherapy, the biocompatibility of nano material is all the time
Being a maximum challenge, platinum cluster has excellent biocompatibility, but up to the present has not yet to see use platinum cluster
Carry out the imaging of tumor and reported by the light heating therapy of Imaging Guidance etc..
Summary of the invention
For the defect of currently available technology, the invention provides an a kind of quick step based on reductive glutathione and close
Become the preparation method and applications of platinum fluorescence nano bunch.Glutathion is wrapped in platinum cluster surface and significantly reduces cell toxicant
Property, under ultraviolet lighting, tumor locus can be carried out fast imaging detection, under infrared light heat effect, tumor can be killed again
Cell.
Technical scheme is:
The preparation method of platinum cluster, comprises the steps: reductive glutathione (GSH) aqueous solution and ascorbic acid
(vitamin C, VC) aqueous solution, adds platinum salt aqueous solution, mix homogeneously, reacts and get final product.
Preferably, described platinum salt is chloroplatinic acid, potassium chloroplatinate, potassium chloroplatinite, ammonium chloroplatinite, ammonium chloroplatinate, chlorine Asia
The mixing of one or any two kinds in potassium platinate, platinum nitrate, tetraammineplatinum chloride, dinitroso diammonia platinum.
Preferably, the mol ratio between reductive glutathione, ascorbic acid and platinum salt is (1～2): (1～2): 1.
Preferably, the concentration of reductive glutathione aqueous solution is 0.01～0.1 mol/L；
Preferably, the concentration of aqueous ascorbic acid is 0.01～0.1mol/L；
Preferably, platinum salt concentration of aqueous solution is 0.01～0.1mol/L；
Preferably, stop when reaction carries out becoming orange to solution；
Preferably, reaction temperature is 5～80 DEG C.
Another aspect, the invention provides the application in the preparation of tumor cell of the above-mentioned platinum cluster；
Another aspect, the invention provides the application in the apoptosis agent of tumor cell of the above-mentioned platinum cluster.
Described tumor cell can be the tumor cell lines such as hepatocarcinoma, pulmonary carcinoma, cervical cancer, leukemia, osteosarcoma.
Beneficial effect
1. the present invention utilizes the synergism of ascorbic acid (vitamin C) and reductive glutathione, and combines gluathione
The corrasion of sulfydryl on peptide, establishes the preparation side of quick one-step synthesis platinum fluorescence nano bunch based on reductive glutathione
Method, the wherein reduction that there was added beneficially reductive glutathione of ascorbic acid.
2. the glutathion that the present invention uses is required composition in target cell, and platinum cluster has wrapped up this kind of glutathion can
To greatly reduce cytotoxicity and to strengthen its targeting.
3. present invention achieves the accurate fast imaging of tumor based on platinum cluster and by the light heating therapy of Imaging Guidance, for
The disease early discoverys such as cancer and treatment provide new method and foundation.
4. this invention can realize not damaged, in situ, Real-time and Dynamic neoplasm targeted therapy, further combined with fluorescence, Raman, super
Sound, CT and nuclear-magnetism etc., can carry out polymorphic with multi-modal synchronization diagnosis and accurately targeting location and treatment, have wide doctor
Learn application prospect.
5. present invention discover that and establish the new of quick one-step synthesis platinum fluorescence nano bunch based on reductive glutathione
Method, and apply to the fast imaging of tumor and by the light heating therapy of Imaging Guidance, for disease early discovery and treatments such as cancers
Provide new method and foundation.
Accompanying drawing explanation
Fig. 1 is the fluorescence spectrum figure in embodiment 1 after platinum cluster synthesis；
Fig. 2 is that in embodiment 1 reference examples, the synthesis of platinum cluster is affected by the ascorbic acid of different amounts.
Fig. 3 a-Fig. 3 c is embodiment 7 fluorescence tomoscan photo, and including matched group and experimental group, wherein, Fig. 3 a is to be not added with
The photo of the hepatoma carcinoma cell of platinum cluster；Fig. 3 b is the photo of the hepatoma carcinoma cell adding platinum cluster；Fig. 3 c adds platinum cluster
The photo of normal cell (L02).
Detailed description of the invention
The reductive glutathione of this experiment, ascorbic acid, chloroplatinic acid, potassium chloroplatinate, potassium chloroplatinite, chloroplatinous acid
The reagent such as ammonium, ammonium chloroplatinate, potassium chloroplatinite are purchased from Chemical Reagent Co., Ltd., Sinopharm Group.Tumor cell used by this test
For tumor cell lines such as hepatocarcinoma, pulmonary carcinoma, cervical cancer, leukemia, osteosarcoma.
The preparation method of embodiment 1 one kinds quick one-step synthesis platinum fluorescence nano bunch based on reductive glutathione:
1) preparation reductive glutathione (GSH) and platinum salt aqueous solution (using chloroplatinic acid in the present embodiment).
2) the reductive glutathione aqueous solution that concentration is 0.05 mol/L (is tieed up raw with the ascorbic acid of 0.05mol/L
Element C) aqueous solution, add the platinum reagent water solution that concentration is 0.05mol/L, the volume ratio of three kinds of solution is 1:1:1, fills
Dividing mixing, then water-bath 37 DEG C heating, reacted when solution becomes orange.
The platinum fluorescence nano bunch good stability uniform particle diameter about 1.2～2.0nm prepared in the present embodiment.
The fluorescence spectrum figure of the platinum fluorescence nano bunch that the present embodiment prepares as it is shown in figure 1, in figure curve 1 be platinum nanometer
Bunch fluorescent spectrum curve, it is bent with the fluorescence spectrum of the mixed solution of ascorbic acid that curve 2 is reductive glutathione (GSH)
Line, 3 is the fluorescent spectrum curve of platinum acid chloride solution, and it is left at 456nm that the most made platinum cluster launches wavelength
The right side, with the Pt of document report0Absorbing wavelength consistent.
Reference examples changes the building-up process of ascorbic acid content in component
Difference with embodiment 1 is: during synthesis, is not added with or to add the ascorbic acid of different content molten
Liquid, as follows:
1) reductive glutathione of different proportion, ascorbic acid and platinum reagent water solution are prepared.
2) be separately added in the chloroplatinic acid aqueous solution that concentration is 0.05mol/L variable concentrations reductive glutathione and
Aqueous ascorbic acid, the volume ratio of three kinds of aqueous solutions is 1:1:1, is sufficiently mixed, and then 37 DEG C of heating in water bath are placed；Carry out altogether
The batch of 5 kinds of variable concentrations ratios, the curve of numbered 1～5 in each batch corresponding diagram 2, concentration is as shown in the table:
(in Fig. 2, the numeral in the legend of each bar curve is the ascorbic acid of the amount mark of the different material after having reacted
Represent the relative amount of ascorbic acid in glutathion with ascorbic acid mole summation) on platinum cluster synthesis impact figure such as
Shown in Fig. 2, as can be seen from the figure do not have glutathion or ascorbic acid all can not obtain platinum fluorescence nano bunch.With embodiment
1 contrast, it can be seen that ascorbic acid serves preferable reduction process synergism, makes reaction be normally carried out.Curve 3 right
The proportioning raw materials answered is optimum, and the fluorescence intensity obtaining product is maximum.
The preparation method of embodiment 2 one kinds quick one-step synthesis platinum fluorescence nano bunch based on reductive glutathione:
1) preparation reductive glutathione (GSH) and platinum salt aqueous solution (using potassium chloroplatinite in the present embodiment).
2) by the ascorbic acid (vitamin of reductive glutathione aqueous solution that concentration is 0.1 mol/L with 0.01mol/L
C) aqueous solution, adds the platinum reagent water solution that concentration is 0.05mol/L, and the volume ratio of three kinds of solution is 2:2:1, three
Being sufficiently mixed, then water-bath 37 DEG C heating, reacted when solution becomes orange.
The platinum fluorescence nano bunch good stability uniform particle diameter about 1.4～2.2nm prepared in the present embodiment.
The transmitting wavelength of the platinum fluorescence nano bunch that the present embodiment prepares is at about 455nm, with the Pt of document report0's
Absorbing wavelength is consistent.
The preparation method of embodiment 3 one kinds quick one-step synthesis platinum fluorescence nano bunch based on reductive glutathione:
1) preparation reductive glutathione (GSH) and platinum salt aqueous solution (using ammonium chloroplatinite in the present embodiment).
2) by the ascorbic acid (vitamin of reductive glutathione aqueous solution that concentration is 0.01 mol/L with 0.1mol/L
C) aqueous solution, adds the platinum reagent water solution that concentration is 0.05mol/L, and the volume ratio of three kinds of solution is 2:1:1, three
Being sufficiently mixed, then water-bath 37 DEG C heating, reacted when solution becomes orange.
The platinum fluorescence nano bunch good stability uniform particle diameter about 1.4～2.3nm prepared in the present embodiment.
The transmitting wavelength of the platinum fluorescence nano bunch that the present embodiment prepares is at about 457nm, with the Pt of document report0's
Absorbing wavelength is consistent.
The preparation method of embodiment 4 one kinds quick one-step synthesis platinum fluorescence nano bunch based on reductive glutathione:
1) preparation reductive glutathione (GSH) and platinum salt aqueous solution (using ammonium chloroplatinate in the present embodiment).
2) the reductive glutathione aqueous solution that concentration is 0.05 mol/L (is tieed up raw with the ascorbic acid of 0.05mol/L
Element C) aqueous solution, add the platinum reagent water solution that concentration is 0.01mol/L, the volume ratio of three kinds of solution is 1:2:1, three
Person is sufficiently mixed, and then water-bath 37 DEG C heating, reacted when solution becomes orange.
The platinum fluorescence nano bunch good stability uniform particle diameter about 1.3～2.1nm prepared in the present embodiment.
The transmitting wavelength of the platinum fluorescence nano bunch that the present embodiment prepares is at about 456nm, with the Pt of document report0's
Absorbing wavelength is consistent.
The preparation method of embodiment 5 one kinds quick one-step synthesis platinum fluorescence nano bunch based on reductive glutathione:
1) preparation reductive glutathione (GSH) and platinum salt aqueous solution (using chloroplatinic acid in the present embodiment).
2) the reductive glutathione aqueous solution that concentration is 0.05 mol/L (is tieed up raw with the ascorbic acid of 0.05mol/L
Element C) aqueous solution, add the platinum reagent water solution that concentration is 0.1mol/L, the volume ratio of three kinds of solution is 1:1:1, three
Person is sufficiently mixed, and then water-bath 37 DEG C heating, reacted when solution becomes orange.
The platinum fluorescence nano bunch good stability uniform particle diameter about 1.1～2.4nm prepared in the present embodiment.
The transmitting wavelength of the platinum fluorescence nano bunch that the present embodiment prepares is at about 457nm, with the Pt of document report0's
Absorbing wavelength is consistent.
The preparation method of embodiment 6 one kinds quick one-step synthesis platinum fluorescence nano bunch based on reductive glutathione:
1) preparation reductive glutathione (GSH) and platinum salt aqueous solution (using chloroplatinic acid in the present embodiment).
2) the reductive glutathione aqueous solution that concentration is 0.05 mol/L (is tieed up raw with the ascorbic acid of 0.05mol/L
Element C) aqueous solution, add the platinum reagent water solution that concentration is 0.05mol/L, three is sufficiently mixed, then water-bath 37
DEG C heating, reacted when solution becomes orange.
The platinum fluorescence nano bunch good stability uniform particle diameter about 1.5～2.5nm prepared in the present embodiment.
The transmitting wavelength of the platinum fluorescence nano bunch that the present embodiment prepares is at about 457nm, with the Pt of document report0's
Absorbing wavelength is consistent.
The accurate fast imaging of embodiment 7 tumor based on platinum cluster
1), after platinum cluster high speed centrifugation embodiment 1 prepared, sterilizing, by ultrasonic dissolution to PBS (pH=
7.2) in case using in, concentration is 100 g/ml.
2) using hepatoma carcinoma cell (HepG2) target cell as object of study, experimental group is by thin for the hepatocarcinoma being in exponential phase
Born of the same parents (HepG2) are according to 1.6 × 105The density of individual cells/well is inoculated in 6 orifice plates, adds step 1) gained after cultivating 24 h
Platinum cluster PBS solution, hatch altogether 12 ~ 72 hours (37 DEG C, 5 % CO2, RH 95%), as experimental group.
3) matched group by hepatoma carcinoma cell (HepG2) target cell in culture medium according to 1.6 × 105The density of individual cells/well
It is inoculated in 6 orifice plates, cultivates 24 h.
4) incubation time terminates every hole addition phosphate buffer solution (PBS, pH=7.2) punching of backward experimental group and matched group
Wash 2-3 time.
5) with confocal fluorescent microscope etc., platinum cluster is carried out qualitative and quantitative analysis.Cell is placed in laser focusing
Under fluorescence microscope, using wavelength is that 405 nm ultraviolet lights carry out exciting the blue-fluorescence image that can collect cell, passes through
Fluorescence layer scanning technology can be concentrated mainly on tumor cell with this platinum nano-cluster visible in detail.Scanned picture
As shown in Fig. 3 a-Fig. 3 c, it can be seen that experimental group (Fig. 3 b) can be transparent to show that out tumor cell, and matched group
(Fig. 3 a) fails demonstrate cellularity；The most in figure 3 c, normal cell is bad with platinum cluster binding ability, it is impossible to aobvious
Image clearly is shown.
Application in the light heating therapy of embodiment 8 tumor based on platinum cluster accurate fast imaging guidance
The platinum cluster that the present invention prepares can in organism some disease site of targeting, such as tumor cell/tissue, it is achieved
To the high time-space resolution fluorescent labeling of the lesions positions such as tumor and by the multi-modal photo-thermal therapy of Imaging Guidance, it is effectively facilitated tumor
Apoptosis and tumor tissues melt.Relevant platinum cluster the imaging of tumor cell and photo-thermal therapy application concrete steps such as
Under:
1), after platinum cluster high speed centrifugation embodiment 1 prepared, sterilizing, by ultrasonic dissolution to PBS (pH=
7.2) in case using in, concentration is 100 g/ml.
2), as experimental group, hepatoma carcinoma cell (HepG2) target cell is selected will to be in logarithm as object of study, experimental group
The hepatoma carcinoma cell (HepG2) of trophophase is according to 1.6 × 105The density of individual cells/well is inoculated in 6 orifice plates, adds after cultivating 24 h
Enter the platinum cluster solution of step 1 gained, after jointly hatching 8 ~ 12 hours, with near infrared light 10 ~ 30 minutes, then followed by
Continuous cultivation 12 ~ 24 hours.
3), as a control group 1, by the hepatoma carcinoma cell (HepG2) in culture medium according to 1.6 × 105The density of individual cells/well
It is inoculated in 6 orifice plates, and experimental group carries out identical incubation time section, with near infrared light 10 ~ 30 minutes, be then further continued for training
Support 12 ~ 24 hours.
4), as a control group 2, by the hepatoma carcinoma cell (HepG2) in culture medium according to 1.6 × 105The density of individual cells/well
It is inoculated in 6 orifice plates, adds the platinum cluster solution of step 1 gained after cultivating 24 h, after jointly hatching 8 ~ 12 hours, with experiment
Unlike group: do not carry out Infrared irradiation, be then cultivated for 12 ~ 24 hours.
5) incubation time terminates every hole addition phosphate buffer solution (PBS, pH=7.2) of backward experimental group and matched group
Rinse 2-3 time, with trypsinization, Trypan Blue, finally by confocal fluorescent microscope observation of cell state, become by fluorescence
The fluorescence distribution situation of picture and its fluorescence intensity carry out qualitative or quantitative analysis to platinum cluster component, and dyeing counting cell withers
The amount of dying.
Data result shows: uses Infrared irradiation to add the apoptosis of tumor cells of this platinum cluster substantially, and is not added with light
According to or add ultraviolet light irradiate cell is had little to no effect.
The above is only the preferred embodiment of the present invention, it should be pointed out that: those skilled in the art are come
Saying, under the premise without departing from the principles of the invention, 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 (1)
1. platinum cluster application in the apoptosis agent preparing tumor cell, it is characterised in that described platinum cluster is to pass through
Following method prepares: is mixed with aqueous ascorbic acid by reductive glutathione aqueous solution, adds platinum salt water-soluble
Liquid, mix homogeneously, react and get final product, wherein, reductive glutathione aqueous solution, aqueous ascorbic acid and the body of platinum salt aqueous solution
Long-pending ratio is 1:1:1；Described platinum salt is chloroplatinic acid；The concentration of reductive glutathione aqueous solution is 0.05 mol/L；Ascorbic acid
The concentration of aqueous solution is 0.05mol/L；Platinum salt concentration of aqueous solution is 0.05mol/L；Reaction temperature is 37 DEG C；Described tumor is thin
Born of the same parents refer to hepatocarcinoma, pulmonary carcinoma, cervical cancer, leukemia or osteosarcoma cell line；The particle size range of the platinum cluster prepared is
1.2～2.0nm.
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| CN106619568B (en) * | 2016-12-09 | 2020-01-17 | 浙江大学 | Preparation method of platinum nanocrystalline composite nanomaterial and product |
| CN108042800B (en) * | 2017-11-16 | 2020-05-26 | 华中科技大学 | Temperature-sensitive polymer modified bivalent platinum nano-cluster and preparation method and application thereof |
| CN108841385B (en) * | 2018-04-27 | 2020-12-25 | 中原工学院 | Preparation method and application of fluorescent platinum nanocluster for optical imaging of blood system suspension cells |
| CN108855240B (en) * | 2018-06-25 | 2020-11-20 | 厦门大学 | Method for protecting catalytic activity of nano platinum particles by using glycerol |
| CN110125434B (en) * | 2019-05-14 | 2022-07-29 | 东南大学 | Preparation method of photo-thermal gold nano material |
| CN110237254B (en) * | 2019-06-05 | 2022-02-15 | 大连工业大学 | Preparation method and application of polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material |
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| EP2226082A2 (en) * | 2009-03-05 | 2010-09-08 | Universität Duisburg-Essen | Control of the toxicity of gold nanoparticles |
| CN103083687A (en) * | 2013-01-16 | 2013-05-08 | 东南大学 | Application of silver and platinum nano-cluster in tumor targeted imaging |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2226082A2 (en) * | 2009-03-05 | 2010-09-08 | Universität Duisburg-Essen | Control of the toxicity of gold nanoparticles |
| CN103083687A (en) * | 2013-01-16 | 2013-05-08 | 东南大学 | Application of silver and platinum nano-cluster in tumor targeted imaging |
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