CN111184875A - A kind of protein/carbon dots nano-hybrid material and its preparation method and application - Google Patents
A kind of protein/carbon dots nano-hybrid material and its preparation method and application Download PDFInfo
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- CN111184875A CN111184875A CN201911293981.2A CN201911293981A CN111184875A CN 111184875 A CN111184875 A CN 111184875A CN 201911293981 A CN201911293981 A CN 201911293981A CN 111184875 A CN111184875 A CN 111184875A
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Abstract
本发明公开了一种蛋白质/碳点纳米杂化材料，其特征在于，所述材料中包含碳点以及与碳点通过共价键偶联的蛋白质。该杂化材料具有稳定的结构，用于制备抗血管栓塞药物时，具有好的溶栓效果及荧光成像效果，进而可通过成像来指示药物的作用部位和代谢部位。本发明还公开了该杂化材料的制备方法和应用。
The invention discloses a protein/carbon point nano hybrid material, which is characterized in that the material comprises carbon points and proteins coupled with the carbon points through covalent bonds. The hybrid material has a stable structure, and when it is used for preparing an anti-vascular embolism drug, it has good thrombolytic effect and fluorescence imaging effect, and can further indicate the action site and the metabolic site of the drug through imaging. The invention also discloses the preparation method and application of the hybrid material.
Description
Technical Field
The invention relates to the technical field of biological imaging materials. More particularly, relates to a protein/carbon dot nano hybrid material, a preparation method and application thereof.
Background
With acute ischemic stroke being a major cause of morbidity and mortality worldwide. Since ischemic stroke is caused by The occlusion of a cerebral artery by a thrombus or embolus, The most reasonable treatment is to remove The occluded artery as soon as possible by thrombolytic therapy and intravascular intervention (The molecular basis of thrombosis and its clinical application stroke. journal of Internal Medicine 2010,267, 191-208). In addition to recombinant tissue plasminogen activator (rt-PA), urokinase-type plasminogen activator (uPA) is the second thrombolytic agent of clinical use, especially in the primary center of stroke (Randomized tertiary of orthogonal infusion of urokinase with 6 routes of midle nuclear biology strain: the midle nuclear biology localized fibrous theory (MELT) Japan Stroke2007,38, 2633-. Venous rt-PA or uPA must be completed within 4.5Hours after onset, and missing the optimal treatment time window may increase complications of cerebral hemorrhage, increasing the risk of morbidity and even mortality (Thrombolysis with Alteplay 3to 4.5.5 Hours after ace approach Ischemic Stroke. New England Journal of medicinal 2008,359, 1317-1329). A large number of animal and clinical imaging studies show that early blood brain barrier injury is closely related to the occurrence of cerebral hemorrhage after thrombolytic reperfusion. Specifically, cerebral ischemia can lead to the rapid loss of local functions of the brain, and cytokines released by astrocytes and endothelial cells can trigger a series of cascade reactions, so that leukocytes are activated. Free radicals and proteolytic enzymes they release can further disrupt the blood brain barrier, leading to blood flow into brain tissue. In addition, uPA is thought to be associated with symptomatic intracranial bleeding, which correlates positively with bleeding rate and thrombolytic agent dose. (symmetric intercarriage of cerebral thrombosis in oral isochaic stroke: assessment of 294 tissues treated with neurological, journal of Neurology, Neurosurgery & Psychiatry 2007,78, 280-285) therefore, by observing the distribution of thrombolytic agents in the brain, early assessment of blood brain barrier injury can greatly improve the probability of detecting hemorrhagic transformation after thrombolysis. Therefore, there is an urgent need to develop an intuitive, sensitive imaging technique based on thrombolytic agents.
Many Imaging techniques have been applied to bioimaging, such as magnetic resonance Imaging (Differential magnetic resonance Types of intramyocardial stress-stress 2015,46,2815-2821), computed tomography (CT contrast fusion: A static phase study of contrast-to-noise ratio and Imaging. journal of Medical Imaging and Radiation Imaging 2017,61,361-366), but these Imaging techniques are usually separate treatment and Imaging in a manner that limits the simultaneous performance of the thrombolytic procedure and Imaging. In addition, radioactive radiation and high costs impose a certain physiological and economic burden on the patient.
Disclosure of Invention
Based on the facts, the first objective of the present invention is to provide a protein/carbon dot hybrid nanomaterial, which has a stable structure, and has better thrombolytic effect and fluorescence imaging effect when being used for preparing anti-vascular-embolism drugs, so that the action site and metabolic site of the drugs can be indicated through imaging.
The second purpose of the invention is to provide a preparation method of the protein/carbon dot nano hybrid material.
The third purpose of the invention is to provide the application of the protein/carbon dot nano hybrid material in preparing the anti-vascular-embolism medicine.
The fourth purpose of the invention is to provide the application of the protein/carbon dot nano hybrid material in fluorescence imaging.
In order to achieve the first purpose, the invention adopts the following technical scheme:
a protein/carbon dot nano hybrid material comprises carbon dots and protein coupled with the carbon dots through covalent bonds.
Further, the carbon dot surface has a reactive group capable of covalently coupling with a protein.
Further, the carbon site is covalently bound to the protein through an amide bond. Compared with physical adsorption, the material has better stability and is less prone to decomposition under complex chemical environment in cells.
Further, the reactive group is one or more of carboxyl, amino, hydroxyl, aldehyde group, epoxy group, mercapto group or isocyanate group.
Further, the particle size of the carbon dots is 1-10nm, and the carbon dots have high fluorescence quantum efficiency.
Further, the material emits fluorescence in the visible to near-infrared regions.
Further, the ultraviolet visible absorption spectrum of the material has a maximum absorption peak between 300-900nm, and the emitted fluorescence is in a visible light to near infrared light region.
Further, the protein is a protein having a thrombolytic effect.
Further, the protein is selected from one of urokinase, tissue plasminogen activator and streptokinase.
Furthermore, the mass ratio of the protein to the carbon dots in the material is 1:1-50:1, preferably 2:1-30: 1.
Further, the preparation of the carbon dots comprises the following steps:
taking a molecule with a reaction group capable of being covalently coupled with protein as a precursor, mixing the precursor with a solvent, carrying out solvothermal reaction in a reaction kettle to obtain a carbon dot crude product, and purifying to obtain the carbon dot.
In order to achieve the second purpose, the invention adopts the following technical scheme:
a preparation method of a protein/carbon dot nano hybrid material comprises the following steps:
activating the reactive groups on the surface of the carbon dots, which can be covalently coupled with protein;
and mixing the activated carbon dot solution with protein, reacting, and performing post-treatment to obtain the protein/carbon dot nano hybrid material.
Further, the coupling agent is selected from one or two of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), Dicyclohexylcarbodiimide (DCC), N-hydroxy thiosuccinimide (sulfo-NHS) and N-hydroxy succinimide (NHS); when the number of the coupling agents is two, the proportion of the two coupling agents is adjusted according to actual needs. Still further, the coupling agent is selected from one of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and Dicyclohexylcarbodiimide (DCC). The coupling agent adopted by the invention has good catalytic activity in a water system, the reaction condition is mild and controllable, and no pollutant is generated.
Further, the temperature of the activation treatment is 25-37 ℃, and the time is 15-30 min.
Further, the reaction temperature is 4-40 ℃, and the reaction time is 24-72 h.
Further, the temperature of the reaction is 25-37 ℃.
Further, the method of activation treatment comprises: fully dissolving the carbon dots with the surface having the reactive groups capable of being covalently coupled with the protein into Phosphate Buffered Saline (PBS) with pH of 7.4 to obtain a carbon dot solution, and adding a coupling agent to activate the reactive groups capable of being covalently coupled with the protein on the surface of the carbon dots.
Further, the post-processing comprises: separating the reacted substance with gel permeation chromatographic column to obtain solution, and freeze drying the solution.
In order to achieve the third purpose, the invention adopts the following technical scheme:
an application of protein/carbon dot nano hybrid material in preparing anti-vascular embolism drugs.
Furthermore, the anti-vascular embolism drugs are anti-cerebral thrombosis drugs, anti-acute myocardial infarction drugs, anti-pulmonary embolism drugs, anti-vascular embolism drugs and anti-myocardial ischemic necrosis drugs.
Further, the application includes:
in order to achieve the fourth object, the invention adopts the following technical scheme:
an application of protein/carbon dot nano hybrid material in fluorescence imaging.
The invention has the following beneficial effects:
the protein/carbon dot nano hybrid material has thrombolytic treatment and fluorescence properties, can be well used for preparing a medicine for simultaneously realizing thrombolytic and fluorescence imaging in cerebral apoplexy diseases, can effectively solve the problem that the existing thrombolytic medicine can only be used for thrombolytic and cannot be imaged, can realize non-invasive fluorescence imaging on the cerebral infarction side in acute ischemic cerebral thrombosis through carbon dots when applied to the acute ischemic cerebral thrombosis, and can indicate the action part and the metabolic part of the medicine through imaging. The hybrid material has excellent performances of both protein and carbon dots, can realize a better thrombolysis effect compared with a single protein, can also realize fluorescence imaging, and can further indicate the action part and the metabolic part of the anti-vascular-embolism medicament through imaging. The lack of any component combination can make the material incapable of having both protein and carbon dot properties.
The preparation method of the protein/carbon dot nano hybrid material is mild and simple, integrates and retains the fluorescence characteristics of the carbon dots and the biological activity of the protein, and realizes the treatment of cerebral thrombosis and fluorescence imaging.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows a transmission electron micrograph of a carbon dot in example 1 of the present invention.
FIG. 2 shows a transmission electron micrograph of a urokinase/carbon dot nanocomposite material in example 1 of the present invention.
FIG. 3 shows Fourier transform infrared spectra of carbon dot, urokinase and urokinase/carbon dot nanocomposites in example 1 of the present invention.
FIG. 4 is a graph showing UV-VIS absorption spectra of a carbon dot, urokinase, and a urokinase/carbon dot nanocomposite material in example 1 of the present invention.
FIG. 5 shows fluorescence emission spectra of the carbon dot and urokinase/carbon dot nanocomposites in example 1 of the present invention.
FIG. 6 shows the fluorescence imaging results of the isolated organs of PBS, urokinase and urokinase/carbon dot nanocomposite material in example 1 of the present invention.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1
The preparation method of the urokinase/carbon dot nano hybrid material comprises the following steps:
1) preparation of carbon dots:
a. weighing 2.0g of citric acid and 938 microliter of ethylenediamine, fully dissolving in 20mL of double distilled water, transferring the solution to a 50mL hydrothermal reaction kettle, reacting at 180 ℃ for 12h, dialyzing the product by using a dialysis bag with molecular weight cutoff of 3500Da, taking out the solution outside the bag, and performing rotary evaporation, concentration and drying;
b. precipitating the dried material with ethanol, freeze drying the precipitate to obtain brown powder, and dispersing 50mg of the brown powder in a solvent containing 2.5g NaOH and 2.5g ClCH2Carrying out ultrasonic treatment for 3h in 50mL aqueous solution of COONa in a dark place, neutralizing with HCl, dialyzing the product by using a dialysis bag with the molecular weight cutoff of 100-plus-500 Da, taking the solution in the bag, and freeze-drying to obtain brown-yellow powder which is carbon dots;
the obtained carbon dots have size of 2-5nm, and have absorption peak at 340nm on ultraviolet visible absorption spectrum and 1730cm on Fourier transform infrared spectrum-1Has a characteristic peak of carboxyl.
2) Preparation of urokinase/carbon dot nano hybrid material:
a. 20mg of the carbon dots were weighed into a 50mL glass vial, and 10mL PBS (0.01mol L) was added-1pH7.4) was sufficiently dissolved to obtain a concentration of 2mg mL-1Carbon dot solution of (5), followed by EDC (400. mu.L, 2mg mL)-1) And NHS (100. mu.L, 2mg mL)-1) Sequentially adding into the solution, and activating at 27 deg.C for 15 min.
b. Adding 50mg of urokinase into the activated carbon dot solution, uniformly mixing, placing the mixture in a shaking table at 37 ℃ for reaction for 24 hours, freeze-drying the solution obtained by separation of a gel permeation chromatographic column to obtain yellow powder, namely the urokinase/carbon dot nano hybrid material, and then placing the yellow powder in a refrigerator at-20 ℃ for storage for later use.
The size of the obtained urokinase/carbon dot nano hybrid material is about 10nm, and absorption peaks at 270nm and 340nm on an ultraviolet visible absorption spectrum respectively exist.
Wherein, fig. 1 is a transmission electron microscopy image of the prepared carbon dot, fig. 3 is a Fourier transform infrared spectrogram of the prepared carbon dot, urokinase and urokinase/carbon dot nano composite material, fig. 4 is an ultraviolet visible absorption spectrogram of the prepared carbon dot, urokinase and urokinase/carbon dot nano composite material, and the prepared carbon dot particles are uniformly distributed and well dispersed and have the size of 2-5nm as shown in fig. 1; 1730cm in the infrared spectrogram (FIG. 3)-1The position represents the existence of carboxyl on the surface of the obtained carbon point; in the ultraviolet visible absorption spectrum (figure 4), two characteristic absorption peaks of carbon points are respectively positioned at 240 nm and 350 nm.
FIG. 2 is a transmission electron micrograph of the urokinase/carbon dot hybrid nanomaterial prepared above, and it can be seen from FIG. 2 that the urokinase/carbon dot hybrid nanomaterial is uniformly distributed, has good dispersibility and has a particle size of about 10 nm; the infrared spectrogram (FIG. 3) converts disappearance of carboxyl peak at carbon point into amido bond (1651 cm) at urokinase/carbon point after the reaction of carboxyl at carbon point with amino on urokinase-1And 1550cm-1). In the ultraviolet visible absorption spectrogram (figure 4), one characteristic peak of urokinase is positioned at 280nm, and two characteristic peaks of urokinase/carbon dot nano hybrid materials are respectively positioned at 270nm and 350 nm. And the complex absorbs more strongly at 300nm or less than the carbon spot due to covalent binding of urokinase to the carbon spot. The above results indicate that urokinase is covalently bound to a carbon site through an amide bond.
Fluorescence characteristics of urokinase/carbon dots: the fluorescence characteristic test of the carbon dot and the urokinase/carbon dot nano hybrid material is completed by a fluorescence spectrophotometer (Cary Eclipse) in a solution state, and the carbon dot and the urokinase/carbon dot are respectively dissolved in PBS (0.01mol L)-1pH7.4) to a final concentration of 0.1mg mL-1The solutions to be tested are placed in cuvettes with light transmission on four sides in sequence and are tested on a fluorescence instrument. And (3) testing temperature: normal temperature; excitation wavelength: 350 nm; sweeping speed: 600 nm/min; slit width: 5.0 nm.
As can be seen from the fluorescence spectrum (figure 5), the urokinase/carbon dot nano hybrid material basically retains the fluorescence property similar to that of the carbon dot, and also emits macroscopic blue fluorescence under the irradiation of an ultraviolet lamp with the wavelength of 365nm, and the light-emitting peak is 450 nm. This result demonstrates that successful labeling of the carbon dot imparts fluorescence properties to urokinase, enabling its use in bioimaging.
In-vitro thrombolysis performance test of urokinase/carbon dot nano hybrid materials:
preparing thrombus: the specimen suppliers are non-blood patients aged 20-30 years, about 5mL venous blood is extracted from antecubital veins, the venous blood is placed into a red vacuum tube without anticoagulant, and the specimen is placed still for 5hours at room temperature to naturally form thrombus.
In vitro thrombolysis performance test: each thrombus was in the form of a long column of about 3cm, the surface water was blotted by filter paper, the surface water was divided into 3 groups by a knife, each was weighed with an analytical balance having an accuracy of 1mg and the value thereof was recorded as M1The degree of dryness and wetness among the thrombi in each group was as consistent as possible. The 3 thrombi were placed in 5mL centrifuge tubes and divided into uPA-CDs + PBS solution groups. 1mL of the solution was added to each tube and incubated in a 37 ℃ bathtub for 2 h. The thrombus was removed, and the surface was again blotted dry with filter paper and weighed with an analytical balance. Marking as M2. The weight loss marker M3＝M1-M2The thrombolysis rate is M3/M1100%. The higher the thrombolysis rate, the better the thrombolysis performance. The thrombolysis rate of the urokinase/carbon dot nano hybrid material prepared by the embodiment is 98.6%.
Fluorescence imaging test of urokinase/carbon dot nano hybrid material:
preparation of transient cerebral ischemia model: male Balb/c mice weighing 20-22g were selected for surgery, and a 0.22 mm + -0.02 silicone-coated monofilament nylon suture was inserted into the internal carotid artery and advanced about 10-12mm along the bifurcation of the internal carotid artery, blocking the middle cerebral artery.
Fluorescence imaging: urokinase/carbon dot material is injected into tail vein after 3h of ischemia and the thrombus is slightly pulled out, after 30min, organs (brain, heart, liver, spleen, lung and kidney) are dissected and taken out, and the organs are placed in a small animal imager (IVIS Spectrum, Perkinelmer, America) for imaging, and laser with the wavelength of 520nm is used for excitation, and imaging is carried out by collecting fluorescence emission signals of the organs. As can be seen from FIG. 6, the urokinase/carbon dot nano hybrid material prepared by the invention has fluorescence imaging performance, and fluorescence imaging is realized on the cerebral infarction side of the operation group instead of the absence of the operation group, which indicates that the material can be used for indicating the injury of the blood brain barrier.
Example 2
Preparation of urokinase/carbon dot nano hybrid material:
the preparation method is the same as that of example 1, except that the hydrothermal method in the step (1a) is changed into a microwave-assisted method, the microwave power and the microwave time are respectively 700W and 2min, and the carbon dots are prepared.
The size of the obtained carbon dots is 1-5nm, and an absorption peak exists at 350nm on an ultraviolet visible absorption spectrum to emit blue fluorescence.
In-vitro thrombolysis performance test of urokinase/carbon dot nano hybrid materials:
the assay was as in example 1, with an in vitro thrombolysis rate of 98.2% at the urokinase/carbon spot.
Fluorescence imaging test of urokinase/carbon dot nano hybrid material:
the test method is the same as that of example 1, and the fluorescence imaging performance is similar to that of example 1.
Example 3
Preparation of urokinase/carbon dot nano hybrid material:
the preparation method is the same as that of example 1, except that the precursor in the step (1a) is changed into p-phenylenediamine, the purification method is changed into gel chromatography, and the red fluorescent carbon dots are obtained by purification. Ethanol precipitation is not used in step (1 b).
The size of the obtained carbon dots is 1-10nm, and an absorption peak exists at 460nm on an ultraviolet visible absorption spectrum to emit red fluorescence.
In-vitro thrombolysis performance test of urokinase/carbon dot nano hybrid materials:
the assay was as in example 1, with an in vitro thrombolysis rate of 98.4% at the urokinase/carbon spot.
Fluorescence imaging test of urokinase/carbon dot nano hybrid material:
the test method is the same as that of example 1, and the fluorescence imaging performance is similar to that of example 1.
Example 4
Preparation of urokinase/carbon dot nano hybrid material:
the preparation method is the same as example 1, except that the activation temperature is changed to 37 ℃ in step (2a), and the urokinase/carbon dot nano hybrid material is prepared.
The size of the obtained urokinase/carbon dot nano hybrid material is about 10nm, and absorption peaks are respectively arranged at 275nm and 340nm on an ultraviolet visible absorption spectrum.
In-vitro thrombolysis performance test of urokinase/carbon dot nano hybrid materials:
the assay was the same as in example 1, with an in vitro thrombolysis rate of 98.8% at the urokinase/carbon spot.
Fluorescence imaging test of urokinase/carbon dot nano hybrid material:
the test method is the same as that of example 1, and the fluorescence imaging performance is similar to that of example 1.
Example 5
Preparation of urokinase/carbon dot nano hybrid material:
the preparation method is the same as that of example 1, except that the reaction time is prolonged to 72 hours after adding urokinase in the step (2b), and the urokinase/carbon dot nano hybrid material is prepared.
The size of the obtained urokinase/carbon dot nano hybrid material is about 10nm, and absorption peaks at 280nm and 345nm on an ultraviolet visible absorption spectrum respectively exist.
In-vitro thrombolysis performance test of urokinase/carbon dot nano hybrid materials:
the assay was as in example 1, with an in vitro thrombolysis rate of 99.0% at the urokinase/carbon spot.
Fluorescence imaging test of urokinase/carbon dot nano hybrid material:
the test method is the same as that of example 1, and the fluorescence imaging performance is similar to that of example 1.
Example 6
Preparation of urokinase/carbon dot nano hybrid material:
the preparation method was the same as example 1 except that the mass of the carbon dots added in step (2) was changed to 10 mg.
In-vitro thrombolysis performance test of urokinase/carbon dot nano hybrid materials:
the assay was as in example 1, with an in vitro thrombolysis rate of 98.1% at the urokinase/carbon spot.
Fluorescence imaging test of urokinase/carbon dot nano hybrid material:
the test method is the same as that of the example 1, the fluorescence signal is very weak, and the imaging effect is not obvious as that of the example 1.
Example 7
Preparing a recombinant tissue plasminogen activator (rt-PA)/carbon dot nano hybrid material:
the preparation method is the same as example 1, except that in the step (2), the urokinase is replaced by rt-PA, and the rt-PA/carbon dot nano hybrid material is prepared.
The size of the obtained rt-PA/carbon dot nano hybrid material is about 10nm, and absorption peaks respectively exist at 280nm and 340nm on an ultraviolet visible absorption spectrum.
Testing the in vitro thrombolysis performance of the rt-PA/carbon dot nano hybrid material:
the test method is similar to that of example 1, except that urokinase is replaced by rt-PA, and the in vitro thrombolysis performance is similar to that of pure rt-PA, and the conclusion is similar to that of example 1.
Fluorescence imaging test of rt-PA/carbon dot nano hybrid material:
the test method is the same as example 1, except that 1) urokinase is changed to rt-PA, and 2) the distance from the site of rt-PA/carbon after tail vein injection is shortened from 30min to 15 min. The fluorescence imaging results are similar to those of example 1.
Comparative example 1
The preparation method of the urokinase/carbon dot nano hybrid material is the same as the step 2 of the example 1, except that no carbon dot is added in the step 2a, and the activated carbon dot solution is replaced by PBS solution containing EDC and NHS in the step 2 b.
The "urokinase/carbon dot hybrid in vitro thrombolytic performance test" in example 1 was repeated except that the "urokinase/carbon dot hybrid solution" was changed to a "urokinase-containing solution" containing the same amount of urokinase.
The above "urokinase-containing solution" was subjected to an in vitro thrombolysis performance test in the same manner as in example 1, and the thrombolysis rate was 96.1%. The fluorescence imaging test was performed on the above "urokinase-containing solution" in the same manner as in example 1. The results indicate that urokinase alone cannot be used for fluorescence imaging applications.
Comparative example 2
The "urokinase/carbon dot hybrid nanomaterial in vitro thrombolysis performance test" in example 1 was repeated except that the "urokinase/carbon dot-containing hybrid nanomaterial solution" was changed to a "carbon dot-containing solution" containing the same amount of carbon dots.
The in vitro thrombolysis performance test of the above "solution containing carbon dots" was performed in the same manner as in example 1, and the results showed that the thrombolysis rate of the carbon dots was similar to that of PBS, indicating that the carbon dots had substantially no thrombolysis performance.
The fluorescence imaging test was performed on the above "solution containing carbon dots" in the same manner as in example 1. The result shows that the fluorescence imaging result of the carbon dot is similar to that of the urokinase/carbon dot nano hybrid material.
Comparative example 3
The preparation method of the urokinase/carbon dot nano hybrid material is the same as that in the example 1, except that the precursors of the carbon dot in the step 1, namely citric acid and ethylenediamine, are replaced by phenylboronic acid, and the prepared carbon dot surface does not contain carboxyl, amino, hydroxyl, aldehyde group, epoxy group, sulfydryl, isocyanate group and other groups.
The preparation method of the phenylboronic acid carbon dots comprises the following steps: 0.2g of phenylboronic acid was dissolved in 20mL of ultrapure water, 0.1M NaOH was added with stirring to adjust the pH to 9.0, and then bubbling with nitrogen gas for 1 hour to remove dissolved O2. Finally, transferring the solution into a hydrothermal reaction kettle, and reacting for 8 hours at 160 ℃. Dialysis with molecular weight cut-off of 1000DaDialyzing the product in a bag, taking out the solution outside the bag, and performing rotary evaporation, concentration and drying. The obtained carbon dot size is 2.5-6.5nm, the absorption peak is at 230-280 nm on the ultraviolet visible absorption spectrum, and 1343cm on the Fourier transform infrared spectrum-1(B-O)、1187cm-1(B-O-H)、1090cm-1(C-B) characteristic peaks.
The fluorescence imaging test of the urokinase/carbon dot nano hybrid material in the example 1 is repeated, and the result shows that the nano hybrid material has no fluorescence property and cannot perform fluorescence imaging.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
Claims (10)
1. A protein/carbon dot nano hybrid material is characterized in that the material comprises carbon dots and protein coupled with the carbon dots through covalent bonds.
2. The protein/carbon dot nanohybrid material according to claim 1, wherein the carbon dot surface has a reactive group capable of covalently coupling with a protein; preferably, the reactive group is one or more of carboxyl, amino, hydroxyl, aldehyde group, epoxy group, mercapto and isocyanate group;
preferably, the particle size of the carbon dots is 1 to 10 nm.
3. The protein/carbon dot nanohybrid material according to claim 1, wherein the material emits fluorescence in the visible to near-infrared region; preferably, the ultraviolet visible absorption spectrum of the material has a maximum absorption peak between 300-900nm, and the emitted fluorescence is in the visible light to near infrared light region.
4. The protein/carbon dot hybrid nanomaterial according to claim 1, wherein the protein is a protein having a thrombolytic effect; preferably, the protein is selected from one of urokinase, tissue plasminogen activator, and streptokinase.
5. The protein/carbon dot hybrid nanomaterial according to claim 1, wherein the mass ratio of the protein to the carbon dots in the material is 1:1-50:1, preferably 2:1-30: 1.
6. The method for preparing protein/carbon dot hybrid nanomaterial according to any of claims 1-5, comprising the steps of:
activating the reactive groups on the surface of the carbon dots, which can be covalently coupled with protein;
and mixing the activated carbon dot solution with protein, reacting, and performing post-treatment to obtain the protein/carbon dot nano hybrid material.
7. The method according to claim 6, wherein the activation treatment is carried out by mixing the carbon dots with a coupling agent to obtain activated carbon dots;
preferably, the coupling agent is selected from one or two of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, N-hydroxythiosuccinimide and N-hydroxysuccinimide.
Preferably, the temperature of the activation treatment is 25-37 ℃, and the time is 15-30 min;
preferably, the reaction temperature is 4-40 ℃, and the reaction time is 24-72 h;
more preferably, the temperature of the reaction is 25-37 ℃.
8. The use of the protein/carbon dot hybrid nanomaterial as defined in any one of claims 1-5 in the preparation of anti-vascular-embolism drugs.
9. The use of claim 8, wherein the anti-thromboembolic drug is an anti-cerebral thrombosis drug, an anti-acute myocardial infarction drug, an anti-pulmonary embolism drug, an anti-thromboembolic drug, an anti-myocardial ischemic necrosis drug.
10. Use of the protein/carbon dot hybrid nanomaterial of any of claims 1-5 in fluorescence imaging.
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