CN112972690B - Triangular nano-gold-based carrier, preparation method and application - Google Patents
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Abstract
The invention relates to the field of biomedicine, in particular to a triangular nano-gold-based carrier, preparation and application, wherein the nano-carrier comprises triangular nano-gold particles and a calcium carbonate layer coated on the surfaces of the triangular nano-gold particles, the nano-probe comprises the nano-carrier and a photosensitizer, the mass ratio of the photosensitizer to the nano-carrier is 1-2. The diagnosis and treatment agent prepared by the invention has multifunctional diagnosis and treatment effect and cancer targeting property.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a triangular nano-gold-based carrier, a preparation method and application thereof.
Background
Castration-resistant prostate cancer is a chronic disease that urinary surgeons face clinically. Once prostate cancer progresses to a castration-resistant prostate cancer stage, no specific treatment means exists in clinic, and the death rate of the disease is high. At present, the most common chemotherapeutic drug for castration-resistant prostate cancer is docetaxel, but the clinical application of docetaxel is limited due to poor water solubility and lack of tumor targeting. Therefore, researchers need to explore and develop a nano diagnosis and treatment probe with more clinical transformation and application potential.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a triangular-based nanogold carrier, a preparation method and a use thereof, which are used for solving the problems in the prior art.
In a first aspect of the present invention, a nano-carrier is provided, where the nano-carrier includes a gold trigonal nanoparticle and a calcium carbonate layer coated on the surface of the gold trigonal nanoparticle.
In a second aspect of the present invention, there is provided a method for preparing the nanocarrier of the first aspect, wherein the method at least comprises the following steps:
1) The method comprises the steps of mixing cetrorhiza chloride, potassium iodide, chloroauric acid and ascorbic acid in a solution with the pH value of 7.4-9.4, reacting, taking precipitate, and purifying again to obtain triangular nano-gold particles.
2) Mixing the triangular nano gold particles and CaCl 2 And Na 2 CO 3 Mixing, reacting, and taking precipitate to obtain precipitate.
3) And mixing the precipitate with polyethylene glycol, reacting, and taking the precipitate to obtain the nano carrier.
In a third aspect of the present invention, a nanoprobe is provided, where the nanoprobe includes the nanocarrier of the first aspect and a photosensitizer, a mass ratio of the photosensitizer to the nanocarrier is 1 to 2.
In a fourth aspect of the present invention, a diagnostic agent is provided, where the diagnostic agent includes the nanocarrier of the first aspect, a photosensitizer and an anticancer drug, a mass ratio of the photosensitizer to the nanocarrier is 1-2.
In a fifth aspect of the present invention, there is provided a method for preparing the therapeutic agent according to the fourth aspect, wherein the method for preparing the therapeutic agent at least comprises the following steps:
1) Mixing the triangular gold nanoparticles, photosensitizer, anticancer drug and CaCl 2 And Na 2 CO 3 Mixing, and centrifuging to remove supernatant after reaction to obtain primary diagnosis and treatment agent;
2) And mixing the primary diagnosis and treatment agent with polyethylene glycol, and centrifuging to obtain a precipitate to obtain the diagnosis and treatment agent.
According to a sixth aspect of the invention, the use of a diagnostic agent according to the fifth aspect for preparing a product for diagnosing and treating tumors is provided.
As mentioned above, the triangular nano-gold-based carrier, the preparation method and the application have the following beneficial effects:
the invention synthesizes the triangular nano gold for the first time, and skillfully coats the photosensitizer medicament indocyanine green ICG and the chemotherapeutic medicament docetaxel by a method for chemically synthesizing calcium carbonate, so that the diagnosis and treatment agent can responsively degrade a surface calcium carbonate layer in a microenvironment of tumor hemic acid so as to release the antitumor medicament in a targeting manner. The polyethylene glycol is used for carrying out biological organic modification on the surface of the calcium carbonate, so that the biocompatibility is increased, and the diagnosis and treatment capability of the diagnosis and treatment agent is improved.
Drawings
FIG. 1 is a transmission electron micrograph prepared according to the present invention.
Fig. 2 shows the results of DLS particle size analysis.
FIG. 3 shows the thermal decay test results of the triangular nano-Au prepared by the present invention.
FIG. 4 shows the results of the photothermal conversion efficiency test of the triangular nano-gold prepared by the present invention
FIG. 5CCK8 tests the viability of cells incubated with PC3 cells.
Fig. 6 fluorescence imaging of agents.
FIG. 7 photograph of tumor-bearing mice irradiated with NIR light for photothermal therapy.
FIG. 8HE detects tumors after treatment with a diagnostic agent.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Please refer to fig. 1 to 8. It should be noted that the drawings provided in the present embodiment are only for illustrating the basic idea of the present invention, and the components related to the present invention are only shown in the drawings rather than drawn according to the number, shape and size of the components in actual implementation, and the type, quantity and proportion of the components in actual implementation may be changed freely, and the layout of the components may be more complicated.
In a first aspect of the present invention, a nano-carrier is provided, where the nano-carrier includes a triangular nano-gold particle and a calcium carbonate layer coated on the surface of the triangular nano-gold particle.
The calcium carbonate layer positioned on the surface of the triangular nano-gold particles can be coated with a medicament, such as a photosensitizer, an anticancer drug and the like, and the calcium carbonate layer is degraded in response in a meta-acid microenvironment to release the coated medicament, so that targeted release can be realized. The triangular nano gold particles have good photo-thermal effect and stability, the photo-thermal conversion efficiency can reach 51.27%, and NIR illumination (1W/cm) is used 2 ) The maximum temperature can reach 70 ℃ in 5min, and after 5 photo-thermal cycles, the maximum temperature can not be obviously reduced.
In one embodiment, the calcium carbonate layer has a thickness of 1 to 5nm.
The thickness of the calcium carbonate layer can be selected by the experimenter according to the requirement of the drug loading, and can be 1-2 nm, 2-3 nm, 3-4 nm or 4-5 nm, for example.
Preferably, the thickness of the calcium carbonate layer is 5nm, and the calcium carbonate layer can cover more medicament.
In one embodiment, the triangular gold nanoparticles have a particle size of 50-65nm.
The particle size of the triangular nano gold particles can be selected by experimenters according to requirements, and can be 50-60 nm or 60-65 nm.
In one embodiment, polyethylene glycol is attached to the surface of the calcium carbonate layer. Such a design is beneficial to increase the biocompatibility of the nanocarrier.
In a second aspect of the present invention, there is provided a method for preparing the nanocarrier of the first aspect, wherein the method at least comprises the following steps:
1) The method comprises the steps of mixing cetrorhiza chloride, potassium iodide, chloroauric acid and ascorbic acid in a solution with the pH value of 7.4-9.4, reacting, taking precipitate, and purifying again to obtain triangular nano-gold particles.
2) Mixing the triangular nano gold particles and CaCl 2 And Na 2 CO 3 Mixing, reacting, and taking precipitate to obtain precipitate.
3) And mixing the precipitate with polyethylene glycol, reacting, and taking the precipitate to obtain the nano carrier.
In the step 1), a certain amount of cetrorhiza chloride, potassium iodide, chloroauric acid and ascorbic acid are weighed and added into an aqueous solution, an alkaline reagent is added to adjust the pH value to be 7.4-9.4, the cetrorhiza chloride, the potassium iodide, the chloroauric acid and the ascorbic acid react for 1-2 hours in the standing process, and after the reaction is finished, the triangular nano-gold particles can be obtained by high-speed centrifugation and purification by using a dialysis membrane.
In the step 2), the triangular nano-gold particles and CaCl obtained in the step 1) are added 2 And Na 2 CO 3 Stirring and mixing, reacting for a period of time, and after the reaction is finished, carrying out high-speed centrifugation by a centrifuge and removing supernatant liquid to obtain a precipitate.
In the step 3), the precipitate obtained in the step 2) is mixed with the activated hyaluronic acid, stirred for a period of time, and centrifuged at a high speed through a centrifuge to obtain the precipitate, so as to obtain the nano-carrier.
In one embodiment, in step 1), the molar ratio of the cetrorhium chloride, the potassium iodide, the chloroauric acid and the ascorbic acid in the solution is 100 to 300:1 to 4.5: 2.5-7.5: 1.5 to 4.8.
In one embodiment, in step 1), the pH of the solution is adjusted with sodium hydroxide.
In one embodiment, in step 1), the rotation speed of the centrifugation is 10000-12000rpm.
In one embodiment, in step 1), dialysis is performed using a dialysis membrane.
In this embodiment, the type of the dialysis membrane: 3500-5000 of MWCO, wherein the dialysis membrane is a conventional product in the market, and is particularly available from specrumlabs company.
In one embodiment, in step 2), the triangular gold nanoparticles and the CaCl are added 2 And said Na 2 The mass ratio of CO is 10:2 to 3:2 to 3.
The experimental personnel select the pentagonal nanogold and the CaCl according to the thickness requirement of the calcium carbonate layer 2 And said Na 2 The mass ratio of CO may be, for example, 10: 2-2.5: 2-2.5, 10: 2.5-3: 2.5-3, 10:2:2 or 10:3:3.
in one embodiment, in step 2), the reaction time is 12 to 36 hours.
The reaction time is chosen by the experimenter and may be, for example, 12 to 24 hours or 24 to 36 hours.
In one embodiment, the mass ratio of the precipitate to the polyethylene glycol is 1 to 3:3 to 5.
The ratio of the mass of the precipitate to the mass of the polyethylene glycol is chosen by the experimenter and may be, for example, 1 to 2: 3-5, 2-3: 3 to 4, 1 to 3:3 to 4 or 1 to 3:4 to 5.
In one embodiment, the time for mixing the precipitate and the polyethylene glycol is 12-18 h.
The time for mixing the precipitate with the activated hyaluronic acid is chosen by the experimenter and may be, for example, 12 to 14 hours, 14 to 16 hours or 16 to 18 hours.
In a third aspect of the present invention, a nanoprobe is provided, wherein the nanoprobe includes the nanocarrier of the first aspect and a photosensitizer, a mass ratio of the photosensitizer to the nanocarrier is 1 to 2.
In this embodiment, the ratio of the photosensitizer to the nanocarrier by mass can be selected by the experimenter, and can be, for example, 1 to 1.5.
In the invention, the photosensitizer is coated in the calcium carbonate layer of the nano-carrier, and the nano-carrier can be oriented to a tumor part to release the photosensitizer, thereby improving the use efficiency of the photosensitizer and reducing the damage of the photosensitizer to normal tissues.
Preferably, the photosensitizer is indocyanine green ICG.
In a fourth aspect, the invention provides a diagnosis and treatment agent, which comprises the nano-carrier, a photosensitizer and an anticancer drug in the first aspect, wherein the mass ratio of the photosensitizer to the nano-carrier is 1-2.
In this embodiment, the experimenter may select the mass ratio of the photosensitizer to the nanocarrier, for example, may be 1 to 1.5.
The diagnosis and treatment agent contains the triangular nano-gold particles, and the calcium carbonate layer can realize responsive degradation of calcium carbonate in a microenvironment with tumor partial acidity due to the characteristics of no toxicity, better biocompatibility, high-efficiency photothermal conversion capability and the like of the triangular nano-gold particles, so that a photosensitizer and an anticancer drug can be released in a targeted manner.
Preferably, the anticancer drug is docetaxel.
In a fifth aspect of the present invention, there is provided a method for preparing the therapeutic agent according to the fourth aspect, wherein the method for preparing the therapeutic agent at least comprises the following steps:
1) Mixing the triangular gold nanoparticles, photosensitizer, anticancer drug and CaCl 2 And Na 2 CO 3 Mixing, and centrifuging to remove supernatant after reaction to obtain primary diagnosis and treatment agent;
2) And mixing the primary diagnosis and treatment agent with polyethylene glycol, and centrifuging to obtain a precipitate to obtain the diagnosis and treatment agent.
In step 1), respectively weighingQuantitative triangular nano gold particles, photosensitizer and anticarcinogen CaCl 2 And Na 2 CO 3 Mixing and stirring for 12-36 h, and after the reaction is finished, centrifuging by using a centrifuge to remove supernatant fluid to obtain the primary diagnosis and treatment agent.
In the step 2), the primary diagnostic agent obtained in the step 1) is mixed with polyethylene glycol, stirred for a period of time, centrifuged at a high speed by a centrifuge, and the centrifuged precipitate is taken to obtain the diagnostic agent.
According to a sixth aspect of the invention, the use of a diagnostic agent according to the fifth aspect for preparing a product for diagnosing and treating tumors is provided.
The diagnosis and treatment agent comprises the triangular nano gold particles and can be used for photothermal treatment of tumors, the photosensitizer and the anticancer drug are coated in the calcium carbonate layer by a method of chemically synthesizing calcium carbonate, the calcium carbonate layer is degraded in a tumor environment, the photosensitizer and the anticancer drug loaded in the cavity are released, targeting release can be realized, and the tumor is treated by combination of chemotherapy, photothermal imaging and fluorescence imaging.
Example 1
(1) 1ml of cetrorhimmonium chloride (CTAC, reagent concentration 0.1M) was diluted with 8ml of ultrapure water, and 100. Mu.l of potassium iodide (KI, reagent concentration 0.01M) and 100. Mu.l of chloroauric acid (HAuCl) were added 4 At a concentration of 25 mM), gently shaken. Then, 20. Mu.l of sodium hydroxide (NaOH, concentration: 0.1M) was added thereto and sufficiently shaken. Then, 50. Mu.l of ascorbic acid (AA, 30mM in concentration) was added thereto and the mixture was thoroughly shaken. Finally, 10. Mu.l of sodium hydroxide (NaOH, concentration 0.1M) is added dropwise again, and the mixture is fully shaken up and then kept stand for 1 to 2 hours. After the reaction is finished, the triangular nano-gold particles can be obtained by high-speed centrifugation (10000-12000 rpm) and purification by using a dialysis membrane.
(2) Weighing 0.5g of triangular nano-gold particles obtained in the steps, and adding the triangular nano-gold particles into 10ml of solution, wherein the solution contains photosensitizer medicine ICG (500 mg/ml) and CaCl 2 Stirring (10 mg/ml) thoroughly for 18-24h, adding DTX (docetaxel) (500 mg/ml) and Na 2 CO 3 (10 mg/ml) solution (10 ml), again stirring well for 24h; after the reaction was completed, the precipitate was obtained by high-speed centrifugation.
(3) Weighing 100mg of the precipitate obtained in the step 2, adding the precipitate into 30ml of ultrapure water, fully and uniformly mixing, adding 300mg of PEG, fully and uniformly mixing and stirring for 12 hours, and removing the supernatant by high-speed centrifugation (8000-12000 rpm) to obtain the diagnosis and treatment agent.
Example 2
(1) 1.5ml of cetrorhiza chloride (CTAC, reagent concentration 0.2M) was diluted with 10ml of ultrapure water, and 150. Mu.l of potassium iodide (KI, reagent concentration 0.03M) and 150. Mu.l of chloroauric acid (HAuCl) were added 4 At a concentration of 50 mM), gently shaken. Then, 50. Mu.l of sodium hydroxide (NaOH, concentration: 0.1M) was added thereto, and the mixture was sufficiently shaken. Then 80. Mu.l of ascorbic acid (AA, 60 mM) was added thereto and the mixture was thoroughly shaken. Finally, 10. Mu.l of sodium hydroxide (NaOH, concentration 0.1M) is added dropwise again, and the mixture is fully shaken up and then kept stand for 1 to 2 hours. After the reaction is finished, the triangular nano gold particles can be obtained by high-speed centrifugation (10000-12000 rpm) and purification by a dialysis membrane.
(2) Weighing 0.5g of triangular nano-gold particles obtained in the steps, and adding the triangular nano-gold particles into 10ml of solution, wherein the solution contains photosensitizer medicine ICG (500 mg/ml) and CaCl 2 Stirring (15 mg/ml) thoroughly for 18-24h, adding DTX (docetaxel) (500 mg/ml) and Na containing chemotherapeutic drug 2 CO 3 (15 mg/ml) solution (10 ml), stirred well again for 36h; after the reaction was completed, the precipitate was obtained by high-speed centrifugation.
(3) Weighing 300mg of the precipitate obtained in the step 2, adding the precipitate into 50ml of ultrapure water, fully and uniformly mixing, adding 300mg of PEG, fully and uniformly mixing and stirring for 12 hours, and removing the supernatant by high-speed centrifugation (8000-12000 rpm) to obtain the diagnosis and treatment agent.
As shown in fig. 1 and 2, the diagnostic agent is observed to be in a regular triangle shape by a transmission electron microscope, and the prepared diagnostic agent is dispersed in distilled water for DLS particle size analysis, and the average diameter of the prepared diagnostic agent is found to be about 80.2nm. The diagnosis and treatment agent is tested by using an atomic absorption spectrum analyzer, and the test result is as follows:
gold element represents gold triangle, calcium element represents calcium carbonate
The multifunctional nanoprobe in the figure is the diagnosis and treatment agent.
Example 3
Adding a certain amount of the diagnosis and treatment agent into distilled water, and uniformly mixing to obtain a mixed solution, wherein the concentration of the triangular nano-gold in the distilled water is 1mg/mL. Using NIR illumination (1W/cm) 2 ) Mixing the solution for 5min, cooling for 5min, repeating for 5 times, and using FLIR TM The E60 camera records the temperature change of the mixed liquor.
As shown in figure 3, the triangular nano gold has good photo-thermal performance, the maximum temperature is about 70 ℃ after 5min of NIR illumination, and after 5 times of thermal cycle, the maximum temperature is not obviously attenuated, and tests prove that the diagnosis and treatment agent has excellent thermal stability.
Example 4
(1) Triangular nano gold with the concentration of 50 mug/ml is measured, and ultrapure water is used as a solvent, and the triangular nano gold is added into a quartz cuvette.
(2) Setting the power of a 808nm laser transmitter to be 2W, enabling the laser ray head to be 2cm away from the sample, and placing a sensor to detect that the output power is about 100mW.
(3) And observing the temperature of the sample by using a thermal infrared imager, and introducing the data into FLIR Tools software in real time by a link computer for analysis and calculation.
As shown in fig. 4, the triangular nano-gold has good photo-thermal performance, and the photo-thermal conversion efficiency is 51.27%.
Example 5
Respectively culturing diagnosis and treatment agents with different concentrations and PC3 cells together to serve as experimental groups, culturing the experimental groups with physiological saline and the PC3 cells together to serve as control groups, incubating for 24h under the environment of cell growth, and staining for CCK8 to calculate the cell activity.
As shown in fig. 5, the cell viability of the diagnosis and treatment agent is not affected, the concentration of the diagnosis and treatment agent is 20 μ g/mL, the cell viability is still greater than 95% when the diagnosis and treatment agent is incubated with PC3 cells, and the biocompatibility of the diagnosis and treatment agent is good.
Example 6
After the PC3 nude mouse tumor-bearing model is successfully established, tail vein is injected with normal saline and the diagnosis and treatment agent (the concentration is 5 mg/mL), and after 48 hours of injection, the Bruker in-vitro fluorescence detection system is used for in vivo observation of the distribution condition of the diagnosis and treatment agent in vivo.
The results are shown in fig. 6, after the injection of the diagnosis and treatment agent, the fluorescence intensity of the tumor part is found to be obviously higher than that of other parts, which indicates that the diagnosis and treatment agent is gathered at the tumor part and can effectively target the tumor.
Example 7
After a PC3 nude mouse tumor-bearing model is successfully established, tail vein is injected with normal saline and a diagnosis and treatment agent (the concentration is 5 mg/mL), and NIR illumination (1W/cm) is used after 48 hours of injection 2 ) The tumor part was irradiated for 3 min. And the recording is shot by a thermal imager.
The results shown in fig. 7 indicate that the temperature of the tumor part was found to be significantly higher than that of other parts after the injection of the diagnostic agent, indicating that the diagnostic agent has a good photothermal effect in the tumor and photothermal treatment ability.
Example 8
Nude mice were divided into two groups, and the diagnosis and treatment agent and physiological saline were injected into the tail vein, respectively. After the in vitro laser excitation treatment, the mice were euthanized for 21 days after continued feeding to obtain tumors, and then standard H & E staining was performed. H & E stain images were recorded using a microscope.
The results are shown in fig. 8, compared with the control group, the nude mice injected with the diagnosis and treatment agent have more tumor necrotic tissues, and the diagnosis and treatment agent prepared by the invention is further proved to have better tumor treatment characteristics.
In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and scope of the present invention as defined in the appended claims.
Claims (5)
1. A diagnostic agent characterized by: the diagnosis and treatment agent comprises a nano-carrier, a photosensitizer and an anticancer drug, wherein the nano-carrier comprises triangular nano-gold particles and a calcium carbonate layer coated on the surfaces of the triangular nano-gold particles, polyethylene glycol is attached to the surface of the calcium carbonate layer, the photosensitizer is indocyanine green, the anticancer drug is docetaxel, the mass ratio of the photosensitizer to the nano-carrier is 1-2.
2. The diagnostic and therapeutic agent according to claim 1, wherein: the thickness of the calcium carbonate layer is 1-5nm.
3. The diagnostic and therapeutic agent according to claim 1, wherein: the triangular nano-gold particles have the particle size of 50-65nm.
4. A method for preparing the diagnostic or therapeutic agent according to any one of claims 1 to 3, comprising at least the steps of:
mixing the triangular gold nanoparticles, photosensitizer, anticancer drug and CaCl 2 And Na 2 CO 3 Mixing, and centrifuging to remove supernatant after reaction to obtain primary diagnosis and treatment agent;
and mixing the primary diagnosis and treatment agent with polyethylene glycol, and centrifuging to obtain a precipitate to obtain the diagnosis and treatment agent.
5. Use of the agent according to any one of claims 1 to 3 for the preparation of a product for the diagnosis and treatment of prostate cancer.
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