CN108578713B - Tumor targeting fluorophore based on folic acid and preparation method and application thereof - Google Patents
Tumor targeting fluorophore based on folic acid and preparation method and application thereof Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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Abstract
The invention provides a preparation method of a tumor targeting fluorophore with ultra-stability and high luminous efficiency based on folic acid, which comprises the following steps: preparing folic acid or a folic acid mixed solution, wherein the folic acid mixed solution consists of folic acid and sugar, citric acid or tris (hydroxymethyl) aminomethane; preparing a sodium hydroxide solution or a potassium hydroxide solution; adding the sodium hydroxide or potassium hydroxide solution into the folic acid or folic acid mixed solution to adjust the pH of the folic acid or folic acid mixed solution to 6-8; and heating to obtain the tumor targeted fluorophore. The method is simple to operate, the reaction process is green and environment-friendly, the obtained fluorophore has high luminous efficiency, good stability and no biotoxicity, has good target combination characteristics on tumors, and has great application potential in the fields of super-resolution fluorescence imaging, target tumor identification, diagnosis and treatment biomedicine and the like; meanwhile, the method has a good application prospect in the aspect of improving the light energy conversion efficiency of solar cells and LEDs.
Description
Technical Field
The invention relates to a preparation method in the field of fluorescent material synthesis, in particular to a folate-based tumor targeting fluorophore with ultra-stability and high luminous efficiency, and a preparation method and application thereof.
Background
The fluorophore has good application prospect in the aspects of biological markers, biomedicine, solar cell conversion efficiency and the like. The traditional organic fluorescent dye has poor stability and is easy to generate photocatalytic degradation; the semiconductor quantum dot material has good light stability and high fluorescence yield. However, it has poor solubility in aqueous solution, and contains heavy metal elements such as Se, Te, Cd, etc., which cause serious damage to human health and environmental pollution. This severely limits its range of applications.
Folic acid (vitamin B9) has good affinity (K) for tumor cells and tissuesd0.1-1 nM) and is widely applied to targeted modification of antitumor drugs. However, as a conventional organic fluorescent dye, it has a series of disadvantages. For example:
1, the fluorescence quantum efficiency is low (less than 0.5 percent in acid, alkaline and neutral environments);
2 solubility was poor, and the solubility in water at 25 ℃ was only 1.6. mu.g.mL-1；
3 poor stability, i.e., easy decomposition under illumination (ultraviolet power of 1 mW. cm)-2Light irradiation for 1h, degradation for 80%), thermal instability (100 ℃ in water solution, heating for 1h, degradation for 40%), and the like.
These disadvantages mentioned above hinder their practical application.
Through retrieval, the Chinese patent application number is CN201710111439.5, which discloses a folic acid-chitosan-Cy 7 polymer with tumor targeting and a preparation method thereof. However, the compound needs to be shielded from light and protected by nitrogen in the process of folic acid targeted modification, and other organic dye molecules need to be coupled to realize selective fluorescent labeling on tumor cells. At the same time, the compound was administered at low drug concentration (40. mu.g.mL)-1) Still exhibit biological cytotoxicity (cell viability less than 50%).
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method of a tumor targeting fluorophore with ultra-stability and high luminous efficiency based on folic acid.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of a tumor targeting fluorophore based on folic acid, which comprises the following steps:
preparing folic acid or a folic acid mixed solution, wherein the folic acid mixed solution consists of folic acid and sugar, citric acid or tris (hydroxymethyl) aminomethane;
preparing a sodium hydroxide solution or a potassium hydroxide solution;
adding the sodium hydroxide or potassium hydroxide solution into the folic acid or folic acid mixed solution to adjust the pH of the folic acid or folic acid mixed solution to 6-8;
and heating the mixed solution after the pH value is adjusted to obtain the tumor targeted fluorophore.
Preferably, the preparation of the folic acid or the folic acid mixed solution specifically comprises: weighing folic acid or folic acid and the saccharide, citric acid or trihydroxymethyl aminomethane, pouring into a container, adding ultrapure water, and performing ultrasonic treatment to obtain yellow suspension.
Preferably, the preparing sodium hydroxide solution or potassium hydroxide solution specifically comprises: weighing sodium hydroxide or potassium hydroxide, pouring into a container, adding ultrapure water, and completely dissolving sodium hydroxide or potassium hydroxide in ultrapure water to obtain colorless, clear and transparent solution.
More preferably, the concentration of the sodium hydroxide or potassium hydroxide solution is not higher than 0.1mmol/mL-1。
Preferably, the pH of the folic acid or the folic acid mixed solution is adjusted to be 6-8, and the method specifically comprises the following steps: adding sodium hydroxide or potassium hydroxide solution into folic acid or folic acid mixed solution in an ultrasonic environment until the solution is converted into clear solution from mixing, and stopping adding the sodium hydroxide or potassium hydroxide solution.
Preferably, the heating treatment of the mixed solution after the pH adjustment is specifically:
(1) placing folic acid or a folic acid mixed solution with the pH value of 6-8 in a reaction kettle;
(2) and (3) placing the reaction kettle in a drying oven, and heating the reaction kettle for 6-8 hours at the constant temperature of 220-250 ℃.
Preferably, the method further comprises, after the heating of the reaction kettle is finished:
(1) centrifugally separating a product obtained by the reaction;
(2) filtering the centrifuged clear solution by using an aqueous ultrafiltration membrane;
(3) freeze drying the filtered solution, dissolving in ultrapure water, and storing at normal temperature for later use.
More preferably, the aqueous ultrafiltration membrane filtration is an aqueous ultrafiltration membrane filtration with a pore size of 220 nm.
The invention also provides a tumor targeting fluorophore based on folic acid, which is obtained by the preparation method.
The invention also provides application of the folate-based tumor targeting fluorophore, wherein the fluorophore realizes specific binding to tumor cells through an endocytosis mechanism mediated by folate receptors on the surfaces of the tumor cells, and is applied to tumor targeting fluorescent labeling, super-resolution imaging, targeted drug delivery or targeted cancer treatment kits. The tumor can include, but is not limited to, breast cancer cells MCF7, breast cancer cells MDA-MB-231, ovarian cancer cells SKOV3, and the like.
In the invention, the fluorescence quantum efficiency of the obtained fluorophore is 60-96%, the preparation method is simple and easy to realize, the prepared fluorophore has high solubility, and the solubility of the fluorophore in water at 25 ℃ can reach 103μg·mL-1Good stability, e.g. 90.81mW cm of light power-2Or the long-term stability is kept in the environment with the temperature of 0-100 ℃, and the tumor targeting property and the biocompatibility are good (the concentration is 10℃)3μg·mL-1Cell viability greater than 98%).
The fluorophore of the present invention is synthesized from folic acid by heating in a neutral environment. Compared with the prior art, the invention has the following beneficial effects:
the invention is simple and economic, is easy to operate, and the prepared fluorophore has high fluorescence quantum yield, can convert ultraviolet light into blue visible light, and has great application prospect in the aspect of improving the light energy conversion efficiency of solar cells and LEDs.
Meanwhile, the material obtained by the invention has good stability, high fluorescence efficiency, no biotoxicity, tumor targeting property and great application potential in the fields of super-resolution fluorescence imaging, targeted tumor identification, diagnosis and treatment biomedicine and the like.
Drawings
FIG. 1 shows the preparation of folate receptor targeting fluorophores and the selective labeling of breast cancer cells MCF7 according to one embodiment of the present invention;
FIG. 2 shows the preparation of folate receptor targeted fluorophores and the MDA-MB-231 selection marker for breast cancer cells according to one embodiment of the present invention;
FIG. 3 shows the preparation of folate receptor-targeted fluorophores and selectable markers for ovarian cancer SKOV3 in accordance with one embodiment of the present invention.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
Example 1 preparation of folate receptor-targeting fluorophore and MCF7 selectable marker for Breast cancer cells
Cleaning 2 100mL beakers in ultrapure water for 30min, and drying for later use after cleaning; sucking 15mL of ultrapure water by using a 5mL liquid-transferring gun, adding the ultrapure water into a beaker, weighing 294mg of folic acid by using an electronic balance, adding the folic acid into the beaker, uniformly stirring by using a glass rod, and carrying out ultrasonic treatment for 5-10 min to obtain yellow suspension; weighing 160mg of sodium hydroxide by using an electronic balance, pouring the sodium hydroxide into another 100mL beaker, and adding 40mL of ultrapure water to completely dissolve the sodium hydroxide in the ultrapure water to obtain a colorless, clear and transparent solution; in an ultrasonic environment, dropwise adding the prepared sodium hydroxide solution into a beaker containing a folic acid solution by using a 3mL pipette until the solution is clear and transparent; sucking 15mL of the solution by using a 5mL pipette, adding the solution into a Teflon reaction kettle with the capacity of 50mL, and heating for 6h at constant temperature at 220 ℃; after the reaction, the solution was taken out at 6000 r.min-1Centrifuging for 16 min; the supernatant was aspirated, filtered using an aqueous filtration membrane having a pore size of 0.22 μm, and then freeze-dried. After drying, dissolving the mixture in ultrapure water according to a certain concentration for later use.
The strongest fluorescence peak is 395.5nm by the test of a fluorometer; the fluorescence quantum efficiency was 60%. Tested by a photobleaching experiment, the light power of the material isIs 90.81mW cm-2(ultraviolet light 26.8mW cm-2(ii) a Visible light 64.1mW cm-2) The light intensity of the light source is continuously exposed for 6 hours, the fluorescence intensity of the light source is high, and the molecular structure is stable. Through the test of a heating experiment, the fluorescence intensity is kept stable when the fluorescence is continuously heated for 6 hours at the temperature of 70 ℃, 80 ℃ and 90 ℃ in the temperature changing process of the temperature range of 0-100 ℃. The fluorophore can be selectively combined with breast cancer tumor MCF7 cells through an endocytosis mechanism mediated by a folate receptor on the surface of the tumor (shown as (a) - (b) in figure 1) through a cell biological experiment test, and does not show cytotoxicity (shown as (c) - (d) in figure 1).
Example 2 preparation of folate receptor-targeting fluorophore and MCF7 selectable marker for Breast cancer cells
Cleaning 2 100mL beakers in ultrapure water for 30min, and drying for later use after cleaning; 15mL of ultrapure water is sucked by a 5mL pipette and added into a beaker, 294mg of folic acid and 40mg of tris (hydroxymethyl) aminomethane are weighed by an electronic balance and added into the beaker, and are stirred and mixed uniformly by a glass rod. Carrying out ultrasonic treatment on the obtained mixed solution for 5-10 min to obtain yellow suspension; weighing 160mg of sodium hydroxide by using an electronic balance, pouring the sodium hydroxide into another 100mL beaker, and adding 40mL of ultrapure water to completely dissolve the sodium hydroxide in the ultrapure water to obtain a colorless, clear and transparent solution; in an ultrasonic environment, dropwise adding the prepared sodium hydroxide solution into a beaker containing folic acid and tris (hydroxymethyl) aminomethane solution by using a 3mL suction tube until the solution is clear and transparent; sucking 15mL of the solution by using a 5mL pipette, adding the solution into a Teflon reaction kettle with the capacity of 50mL, and heating for 6h at constant temperature at 220 ℃; after the reaction, the solution was taken out at 6000 r.min-1Centrifuging for 16 min; the supernatant was aspirated, filtered using an aqueous filtration membrane having a pore size of 0.22 μm, and then freeze-dried. After drying, dissolving the mixture in ultrapure water according to a certain concentration for later use.
The strongest fluorescence peak is 395.5nm by the test of a fluorometer; the fluorescence quantum efficiency was 77%. The test of a photobleaching experiment shows that the luminous power of the material is 90.81mW cm-2(ultraviolet light 26.8mW cm-2(ii) a Visible light 64.1mW cm-2) The light intensity of the light source is continuously exposed for 6 hours, the fluorescence intensity of the light source is high, and the molecular structure is stable. Through heating experimentTesting, the fluorescence intensity is kept stable when the fluorescent powder is continuously heated for 6 hours at the temperature of 70 ℃, 80 ℃ and 90 ℃ in the temperature changing process of the temperature range of 0-100 ℃. The fluorophore can selectively bind to MDA-MB-231 cells of breast cancer tumor (shown in (a) - (b) in FIG. 2) through an endocytosis mechanism mediated by folate receptor on the surface of the tumor, and does not show cytotoxicity (shown in (c) - (d) in FIG. 2) through tests of cell biology experiments
Example 3 preparation of folate receptor-targeting fluorophore and selectable marker for ovarian cancer cell SKOV3
Cleaning 2 100mL beakers in ultrapure water for 30min, and drying for later use after cleaning; 15mL of ultrapure water is sucked by a 5mL pipette and added into a beaker, 294mg of folic acid and 100mg of fructose are weighed by an electronic balance and added into the beaker, and the mixture is stirred and mixed uniformly by a glass rod. Carrying out ultrasonic treatment on the obtained mixed solution for 5-10 min to obtain yellow suspension; weighing 160mg of potassium hydroxide by using an electronic balance, pouring the potassium hydroxide into another 100mL beaker, and adding 40mL of ultrapure water to completely dissolve the sodium hydroxide in the ultrapure water to obtain a colorless, clear and transparent solution; in an ultrasonic environment, dropwise adding the prepared potassium hydroxide solution into a beaker containing a solution of folic acid and fructose by using a 3mL suction pipe until the solution is clear and transparent; sucking 15mL of the solution by using a 5mL pipette, adding the solution into a Teflon reaction kettle with the capacity of 50mL, and heating for 8h at the constant temperature of 250 ℃; after the reaction, the solution was taken out at 6000 r.min-1Centrifuging for 16 min; the supernatant was aspirated, filtered using an aqueous filtration membrane having a pore size of 0.22 μm, and then freeze-dried. After drying, dissolving the mixture in ultrapure water according to a certain concentration for later use.
The carbon nanoparticles were obtained by transmission electron microscopy analysis, and the particle size was about 5 nm. The strongest fluorescence peak is 395.5nm by the test of a fluorometer; the fluorescence quantum efficiency was 96%. The test of a photobleaching experiment shows that the luminous power of the material is 90.81mW cm-2(ultraviolet light 26.8mW cm-2(ii) a Visible light 64.1mW cm-2) The light intensity of the fluorescent light is kept stable after 6 hours of continuous exposure. The fluorescence intensity is kept stable by the test of heating experiment when the fluorescence is continuously heated for 6h at the temperature of 70 ℃, 80 ℃ and 90 ℃ in the temperature changing process of 0-100 ℃. By cell biologyExperimental tests show that the fluorescent nanoparticle can selectively bind to ovarian cancer SKOV3 cells through an endocytosis mechanism mediated by folate receptors on the surface of tumors (shown as (a) - (b) in fig. 3), and does not show cytotoxicity (shown as (c) - (d) in fig. 3).
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (8)
1. A preparation method of a tumor targeting fluorophore based on folic acid is characterized in that:
preparing a folic acid mixed solution, wherein the folic acid mixed solution consists of folic acid and fructose or tris (hydroxymethyl) aminomethane;
preparing a sodium hydroxide solution or a potassium hydroxide solution;
adding the sodium hydroxide or potassium hydroxide solution into the folic acid mixed solution to adjust the pH of the folic acid mixed solution to 6-8;
the pH value of the folic acid mixed solution is adjusted to be 6-8, and the method specifically comprises the following steps: adding a sodium hydroxide or potassium hydroxide solution into a folic acid mixed solution in an ultrasonic environment until the solution is converted into a clear solution from mixing, and stopping adding the sodium hydroxide or potassium hydroxide solution;
heating the mixed solution after the pH value is adjusted to obtain a tumor targeted fluorophore;
the heating treatment of the mixed solution after the pH value is adjusted specifically comprises the following steps:
(1) taking a folic acid mixed solution, and placing the folic acid mixed solution in a reaction kettle;
(2) and (3) placing the reaction kettle in an oven, and heating the reaction kettle for 6-8 hours at a constant temperature of 220-250 ℃.
2. The method of claim 1, wherein the folate-based tumor-targeted fluorophore is selected from the group consisting of: the preparation of the folic acid mixed solution specifically comprises the following steps: weighing folic acid and the fructose or the trihydroxymethyl aminomethane, pouring the folic acid and the fructose or the trihydroxymethyl aminomethane into a container, adding ultrapure water, and performing ultrasonic treatment to obtain a yellow suspension.
3. The method of claim 1, wherein the folate-based tumor-targeted fluorophore is selected from the group consisting of: the preparation of the sodium hydroxide solution or the potassium hydroxide solution specifically comprises the following steps: weighing sodium hydroxide or potassium hydroxide, pouring into a container, adding ultrapure water, and completely dissolving the sodium hydroxide or potassium hydroxide in the ultrapure water to obtain a colorless, clear and transparent solution.
4. The method of claim 3, wherein the fluorophore is selected from the group consisting of: the concentration of the sodium hydroxide or potassium hydroxide solution is not higher than 0.1 mmol/mL.
5. The method of claim 1, wherein the folate-based tumor-targeted fluorophore is selected from the group consisting of: after the heating of the reaction kettle is finished:
(1) centrifugally separating a product obtained by the reaction;
(2) filtering the centrifuged clear solution by using an aqueous ultrafiltration membrane;
(3) and freeze-drying the filtered solution, dissolving the solution in ultrapure water according to the concentration required by the experiment, and storing the solution at normal temperature for later use.
6. The method of claim 5, wherein the folate-based tumor-targeted fluorophore is selected from the group consisting of: and filtering by using the aqueous ultrafiltration membrane, namely filtering by using the aqueous ultrafiltration membrane with the pore diameter of 220 nm.
7. A folate-based tumor-targeting fluorophore characterized by being obtainable by the preparation process according to any one of claims 1 to 6.
8. A kit for tumor targeting fluorescent labeling, super-resolution imaging, targeted drug delivery or targeted cancer therapy is characterized in that: the folate-based tumor-targeting fluorophore of claim 7, wherein the folate-based tumor-targeting fluorophore specifically binds to tumor cells via an endocytosis mechanism mediated by folate receptors on the surface of the tumor cells.
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| CN102942924A (en) * | 2012-10-22 | 2013-02-27 | 上海交通大学 | Preparation method of carbon quantum dots based on chemical reaction of fructose and sodium hydroxide |
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| Title |
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| Haifang Liu et al..Synthesis of Luminescent Carbon Dots with Ultrahigh Quantum Yield and Inherent Folate Receptor-Positive Cancer Cell Targetability.《Scientific Reports》.2018,第8卷 * |
| Synthesis of Luminescent Carbon Dots with Ultrahigh Quantum Yield and Inherent Folate Receptor-Positive Cancer Cell Targetability;Haifang Liu et al.;《Scientific Reports》;20180118;第8卷;第1086页 * |
| 碳量子点的制备及其在能源与环境领域应用进展;傅鹏等;《应用化学》;20160731;第33卷(第7期);第742-755页 * |
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| CN108578713A (en) | 2018-09-28 |
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