CN102876328A - Near-infrared fluorescent nanoparticle in-vivo probe and preparation method thereof - Google Patents
Near-infrared fluorescent nanoparticle in-vivo probe and preparation method thereof Download PDFInfo
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- CN102876328A CN102876328A CN2012104072993A CN201210407299A CN102876328A CN 102876328 A CN102876328 A CN 102876328A CN 2012104072993 A CN2012104072993 A CN 2012104072993A CN 201210407299 A CN201210407299 A CN 201210407299A CN 102876328 A CN102876328 A CN 102876328A
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Abstract
The invention relates to a near-infrared fluorescent nanoparticle in-vivo probe. The near-infrared fluorescent nanoparticle in-vivo probe comprises water-soluble near-infrared CdHgTe fluorescent quantum dots and liposomal phospholipid film wrapped outside the dots. The invention further provides a preparation method of the near-infrared fluorescent nanoparticle in-vivo probe. The preparation method includes the steps of firstly, synthetizing the water-soluble near-infrared CdHgTe fluorescent quantum dots; secondly, preparing the liposomal phospholipid film; and thirdly, adding water-soluble near-infrared fluorescent quantum dots CdHgTe solution synthetized in the step 1 into the liposomal phospholipid film prepared in the step 2 to obtain the near-infrared fluorescent nanoparticle in-vivo probe. The near-infrared fluorescent nanoparticle in-vivo probe is high in stability and biocompatibility. The preparation process is simple, the cost is low, and the near-infrared fluorescent nanoparticle in-vivo probe is convenient to use and effective in in-vivo imaging.
Description
Technical field
The present invention relates near infrared living imaging field, be specifically related to fluorescence nano grain probe in a kind of near infrared body, simultaneously the present invention also provides the preparation method of fluorescence nano grain probe in a kind of near infrared body.
Background technology
The near infrared imaging technology is a kind of novel light spectrum image-forming means that development in recent years is got up.In the applied near-infrared band of this technology (700-900nm) scope, avoided the main uptake zone of a lot of endogenous material such as aerobic oxyphorase, anaerobic oxyphorase, water, thereby the penetration depth in biological tissue large (can reach 10cm), and this wave band archebiosis fluorescence also is down to minimum degree, and imaging signal is subjected to the impact of biological tissue's background also fewer.The potentiality of near infrared imaging technology harmless continuous monitoring in place biological tissue have begun to obtain to pay attention at the Disciplinary Frontiers of the life sciences such as drug research, tumor diagnosis and therapy.
The research and development of near infrared fluorescent probe are bottleneck problems of near infrared imaging technical development.At present, the probe kind that is fit to be applied to near-infrared region is also very limited, mainly concentrates on some organic fluorescent dyes.But the shortcoming such as this class organic dye ubiquity excitation spectrum is narrow, utilizing emitted light spectrum width and anti-photobleaching are poor.Recently, near infrared fluorescence quantum point such as CdHgTe, CdTeSe/CdS have caused people's very big interest and concern.The luminous wide coverage of this class inorganic nano fluorescent material, spectrum is easy to tuning, and fluorescence efficiency is also higher, as a kind of novel near infrared fluorescent probe, has huge application potential in the living body biological imaging research.Yet independent common near-infrared quantum dots is also having some problems to overcome aspect biocompatibility, stability, the toxicity at present.
For the stability of improving common quantum dot and the problem of toxicity, usually the mode that adopts is that quantum dot is carried out finishing, preparation functional quantum point or it is wrapped in the polymer microsphere, but these methods tend to reduce fluorescence intensity or the body internal stability of quantum dot, some polymer microsphere is because volume is excessive and be not suitable for in-vivo imaging, and they also can need to use some expensive reagent or very harsh reaction conditions.Therefore, demand urgently developing a kind ofly new have good biocompatibility, stability, modifying method that toxicity is low wraps up independent quantum dot, to realize that it in vivo, have better application in the outer imaging.
Summary of the invention
The technical problem that the present invention solves is to have overcome the deficiency of near-infrared quantum dots aspect stability, biocompatibility independent in the prior art, and fluorescence nano grain probe in a kind of near infrared body is provided.
For achieving the above object, fluorescence nano grain probe in the near infrared body of the present invention comprises the liposome immobilized artificial membrane of water-soluble near infrared CdHgTe fluorescence quantum and its coated outside.
Simultaneously, the invention provides the preparation method of fluorescence nano grain probe in a kind of near infrared body, the method comprises the steps:
Step 2, preparation liposome immobilized artificial membrane;
Step 3, the aqueous solution of the water-soluble near infrared CdHgTe fluorescence quantum that step 1 is synthetic joins in the liposome immobilized artificial membrane of step 2 preparation and carries out hydration, obtain fluorescence nano grain probe, i.e. near infrared CdHgTe quantum dot-liposome fluorescent probe in the near infrared body.
As the restriction to aforesaid way, the process of the water-soluble near infrared CdHgTe of step 1 fluorescence quantum is with NaHTe solution, Cd (NO
3)
2Solution and Mercury pernitrate solution are under the effect of Thiovanic acid (MPA), according to Cd
2+: the mol ratio of NaHTe: MPA is to react at 1: 0.5: 2.4 to obtain.
As the further restriction to aforesaid way, described NaHTe solution prepares by the following method: with tellurium powder and NaBH
4According to 3: 2 mass ratio, at N
2The gas protection is lower to add distilled water, reacts in 35-45 ℃ of water-bath, obtains NaHTe solution.
As the further restriction to aforesaid way, described step 2 is Yelkin TTS, cholesterol and PEG-6000 to be dissolved in chloroform obtain mixing solutions, after this solution decompression is concentrated, obtains the liposome immobilized artificial membrane.
As the further restriction to aforesaid way, described concentrating under reduced pressure is to carry out under constant temperature.
In addition, as the restriction to aforesaid way, step 3 with the hydro-combination process of water-soluble near infrared CdHgTe fluorescence quantum and liposome immobilized artificial membrane is: the aqueous solution of water-soluble near infrared CdHgTe fluorescence quantum is joined in the liposome immobilized artificial membrane, and film is washed in jolting; After liposome membrane hydration to be formed becomes the emulsus liposome turbid liquor, under condition of ice bath, carry out ultra-sonic dispersion, obtain fluorescence nano grain probe in the near infrared body.
Main contributions of the present invention is the thinking of fluorescent probe preparation, and the reagent that the present invention is used and processing unit are in the prior art commonly use, are not key of the present invention, therefore simple description.
Beneficial effect of the present invention is as follows:
The method is synthesizing water-solubility near infrared CdHgTe fluorescence quantum at first, then it is wrapped in the homemade liposome water cavity, through freezing ultrasonic, obtains the near infrared living body fluorescent probe that spectrum property is stable, biocompatibility is good.Its preparation process is simple, and low cost is easy to use, and in-vivo imaging is respond well.
Description of drawings
Below in conjunction with the drawings and the specific embodiments the present invention is done further detailed description:
Fig. 1 is the fluorescence spectrum figure of near infrared CdHgTe quantum dot-liposome fluorescent probe.
Fig. 2 is near infrared CdHgTe quantum dot-liposome fluorescent probe transmission electron microscope picture.
Fig. 3 is independent CdHgTe quantum dot and the anti-photobleaching experimental result picture of near infrared CdHgTe quantum dot-liposome fluorescent probe.
Fig. 4 is the independent CdHgTe quantum dot of three kinds of different concns and the cell viability experimental result picture of near infrared CdHgTe quantum dot-liposome fluorescent probe.As seen the cytotoxicity of the prepared fluorescent probe of the present invention all significantly is lower than independent CdHgTe quantum dot under three kinds of different concns.
Fig. 5 is the living imaging figure of near infrared CdHgTe quantum dot-liposome fluorescent probe in Mice Body.
Embodiment
The below describes raw material sources and the instrument model of using among the embodiment:
Cd (NO
3)
22.5H
2O, tellurium powder, dimethyl sulfoxide (DMSO) (DMSO), NaBH
4Be purchased from Shanghai reagent company.PEG-6000, Thiovanic acid (MPA) are purchased from Shanghai Ling Feng reagent company.Ovum Gallus domesticus Flavus lecithin (lipoid, Germany) is available from the too big medicine company in Shanghai, and cholesterol, chloroform are available from Jiangsu magnificent fine chemicals company limited forever.Tetramethyl-azo azoles salt (MTT) is available from Sigma company.All cells is cultivated reagent and all is purchased from U.S. Gibico.
The used spectrograph of Fig. 1, Fig. 3 is RF-5301 type spectrophotofluorometer, Japanese Shimadzu company.
The used transmission electron microscope of Fig. 2 is JEM-2100 type electron microscope, Jeol Ltd..
The used enzyme connection instrument of Fig. 4 is 1500 type enzyme immunoassay instrument, U.S. Thermo Electron company.
Laser apparatus is HLU32F400808nm in the used near infrared imaging system of Fig. 5, German Limo company.
The reagent that the present invention is used and processing unit are in the prior art to be commonly used, and are not key of the present invention, therefore simple the description.
Embodiment one
Step 1: the preparation of water-soluble near infrared CdHgTe fluorescence quantum
Precision takes by weighing tellurium powder 60mg, NaBH
440mg places the three-necked bottle of 25ml, at N
2The gas protection is lower to add redistilled water 2ml, behind the reaction 30min, obtains red-purple NaHTe solution in 40 ℃ of water-baths.In this solution, pass into N
2Gas 30min is with this solution deoxidation.Other prepares 100ml and contains 92.4mgCd (NO
3)
2Solution, and to add 63 μ l Thiovanic acids (MPA) be stablizer, transfers pH to 11.2 with 1mol/L NaOH, to wherein adding 10 μ l Mercury pernitrate solution.In this solution, pass into N
2Gas 30min is with this solution deoxidation.Under violent stirring, inject the 0.5mol/L NaHTe solution 600 μ l of above-mentioned deoxidation, make Cd
2+: the mol ratio of NaHTe: MPA is 1: 0.5: 2.4, then rapidly extremely boiling of reflux.Refluxed 1 hour, and obtained CdHgTe solution.
Step 2: the preparation of liposome immobilized artificial membrane
Yelkin TTS 25mg, cholesterol 3mg, PEG-600040mg be dissolved in the 5ml chloroform obtain solution, shift the chloroformic solution of above-mentioned 5mL in the 500mL eggplant-shape bottle, 37 ℃ of waters bath with thermostatic control, rotary evaporation is 15 minutes on Rotary Evaporators, remove organic solvent, make it form even class membrane of lipoprotein in flask walls.
Step 3: the preparation of CdHgTe quantum dot-liposome fluorescent probe
In flask, pass into N
2Carry out deoxygenation 5min, treat that solvent removes fully, add the CdHgTe solution of 2mL above-mentioned steps one preparation, firmly film is washed in jolting.After lipoid film hydration to be formed became the emulsus liposome turbid liquor, ice-bath ultrasonic was disperseed this suspension, namely gets near infrared CdHgTe quantum dot-liposome fluorescent probe.The near infrared CdHgTe quantum dot that this step is obtained-liposome fluorescent probe carries out fluorescent spectroscopy, carries out spectral scan with spectrophotofluorometer, the results are shown in Figure 1; Be added drop-wise to after the dilution on the 400 order copper mesh that support carbon film, use transmission electron microscope observing after the drying, the results are shown in Figure 2.
Embodiment two
(1) study on the stability of CdHgTe quantum dot and CdHgTe quantum dot-liposome fluorescent probe:
Get among the embodiment one the CdHgTe quantum dot of preparation and CdHgTe quantum dot-liposome fluorescent probe and all place under the 20W254nm ultraviolet lamp and shine, and measure fluorescence intensity, relatively both anti-photobleaching abilities 0,2,4,6,8, during 10h.The results are shown in Figure 3.As seen the light stability of CdHgTe quantum dot-liposome fluorescent probe obviously is better than independent CdHgTe quantum dot.
(2) Toxicity test of CdHgTe quantum dot and CdHgTe quantum dot-liposome fluorescent probe:
Choose human breast cancer cell MCF-7 cell, add in 96 well culture plates, establish five multiple holes, put 37 ℃, 5%CO
2Incubated overnight in the incubator, the M199 nutrient solution of nutrient solution being replaced by 0.5% serum makes cell synchronization, then add CdHgTe quantum dot prepared among the embodiment 1 of high, normal, basic three kinds of concentration and CdHgTe quantum dot-liposome, 37 ℃ hatch 24h after, every hole adds 5mg/ml MTT solution 20 μ l, continue to cultivate 4h, directly discard whole supernatant liquors.Add DMSO100 μ l/ hole, vibrate 5min on the microoscillator, crystallization is dissolved fully, enzyme connection 490nm wavelength place measures absorbance.The results are shown in Figure 4.As seen the cell viability of CdHgTe quantum dot-liposome fluorescent probe all is significantly higher than independent CdHgTe quantum dot under different concns, i.e. the prepared CdHgTe quantum dot of the present invention-liposome cytotoxicity is much smaller than independent CdHgTe quantum dot.
(3) living imaging of near infrared CdHgTe quantum dot-liposome fluorescent probe:
Get near infrared CdHgTe quantum dot-liposome solutions of obtaining among the embodiment 1 with 5 μ g/g tail vein injections in through Na
2In the Mice Body of S depilation (Kunming mouse), the results are shown in Figure 5.As shown in FIG., under the near infrared imaging system, CdHgTe quantum dot-liposome fluorescence nano grain enters in the Mice Body and can present fluorescence.Behind the 10min, visible liver position shows extremely strong fluorescent signal among the shooting gained figure in injecting Mice Body.
Claims (7)
1. fluorescence nano grain probe in the near infrared body, it is characterized in that: it comprises the liposome immobilized artificial membrane of water-soluble near infrared CdHgTe fluorescence quantum and its coated outside.
2. the preparation method of the interior fluorescence nano grain probe of near infrared body is characterized in that the method comprises the steps:
Step 1, synthesizing water-solubility near infrared CdHgTe fluorescence quantum;
Step 2, preparation liposome immobilized artificial membrane;
Step 3, the aqueous solution of the water-soluble near infrared CdHgTe fluorescence quantum that step 1 is synthetic join in the liposome immobilized artificial membrane of step 2 preparation carries out hydration, obtains fluorescence nano grain probe in the near infrared body.
3. the preparation method of fluorescence nano grain probe in the near infrared body according to claim 2, it is characterized in that: the process of the water-soluble near infrared CdHgTe of step 1 fluorescence quantum is with NaHTe solution, Cd (NO
3)
2Solution and Mercury pernitrate solution are under the effect of Thiovanic acid (MPA), according to Cd
2+: the mol ratio of NaHTe: MPA is to react at 1: 0.5: 2.4 to obtain.
4. the preparation method of fluorescence nano grain probe in the near infrared body according to claim 3 is characterized in that described NaHTe solution prepares by the following method: with tellurium powder and NaBH
4According to 3: 2 mass ratio, at N
2The gas protection is lower to add distilled water, reacts in 35-45 ℃ of water-bath, obtains NaHTe solution.
5. the preparation method of fluorescence nano grain probe in each described near infrared body in 4 according to claim 2, it is characterized in that: described step 2 is Yelkin TTS, cholesterol and PEG-6000 to be dissolved in chloroform obtain mixing solutions, after this solution decompression is concentrated, obtain the liposome immobilized artificial membrane.
6. the preparation method of fluorescence nano grain probe in the near infrared body according to claim 5, it is characterized in that: described concentrating under reduced pressure is to carry out under constant temperature.
7. the preparation method of fluorescence nano grain probe in the near infrared body according to claim 2, it is characterized in that, step 3 with the hydro-combination process of water-soluble near infrared CdHgTe fluorescence quantum and liposome immobilized artificial membrane is: the aqueous solution of water-soluble near infrared CdHgTe fluorescence quantum is joined in the liposome immobilized artificial membrane, and film is washed in jolting; After liposome membrane hydration to be formed becomes the emulsus liposome turbid liquor, under condition of ice bath, carry out ultra-sonic dispersion, obtain fluorescence nano grain probe in the near infrared body.
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Cited By (4)
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| CN103552995A (en) * | 2013-09-29 | 2014-02-05 | 苏州大学 | Method for preparing near-infrared quantum dots |
| CN103808699A (en) * | 2012-11-15 | 2014-05-21 | 中国科学院理化技术研究所 | Lipidosome carrying quantum dot and enzyme as well as preparation method and application thereof |
| CN103992798A (en) * | 2014-06-11 | 2014-08-20 | 武汉大学 | Preparation method of CdHgTe alloy quantum dots |
| CN104673316A (en) * | 2013-12-02 | 2015-06-03 | 天津大学 | Tellurium-mercury-cadmium quantum dot/carbon nanotube nano composite material and preparation method thereof |
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| WO2005071115A1 (en) * | 2004-01-21 | 2005-08-04 | Georgia Tech Research Corporation | Activatable probes and methods for in vivo gene detection |
| CN101327189A (en) * | 2008-07-31 | 2008-12-24 | 上海交通大学 | Method for preparing nano alcohol liposome material with mark and treatment double-function |
Non-Patent Citations (3)
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103808699A (en) * | 2012-11-15 | 2014-05-21 | 中国科学院理化技术研究所 | Lipidosome carrying quantum dot and enzyme as well as preparation method and application thereof |
| CN103808699B (en) * | 2012-11-15 | 2016-03-09 | 中国科学院理化技术研究所 | Load liposome of quantum dot and enzyme and its preparation method and application |
| CN103552995A (en) * | 2013-09-29 | 2014-02-05 | 苏州大学 | Method for preparing near-infrared quantum dots |
| CN104673316A (en) * | 2013-12-02 | 2015-06-03 | 天津大学 | Tellurium-mercury-cadmium quantum dot/carbon nanotube nano composite material and preparation method thereof |
| CN103992798A (en) * | 2014-06-11 | 2014-08-20 | 武汉大学 | Preparation method of CdHgTe alloy quantum dots |
| CN103992798B (en) * | 2014-06-11 | 2016-06-22 | 武汉大学 | A kind of preparation method of CdHgTe alloy quantum dot |
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Application publication date: 20130116 |