CN114617963A - Synthesis method of gold nano-drug for targeted killing of tumor cells through cooperation of autophagy inhibition and photothermal therapy - Google Patents

Synthesis method of gold nano-drug for targeted killing of tumor cells through cooperation of autophagy inhibition and photothermal therapy Download PDF

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CN114617963A
CN114617963A CN202210103500.2A CN202210103500A CN114617963A CN 114617963 A CN114617963 A CN 114617963A CN 202210103500 A CN202210103500 A CN 202210103500A CN 114617963 A CN114617963 A CN 114617963A
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李丹
赵亭磊
蒋艳夏蕾
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Linyi University
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Abstract

The invention discloses a synthesis method of a gold nano-drug for inhibiting autophagy and killing tumor cells in a targeted manner by cooperation with photothermal therapy, and belongs to the technical field of drug synthesis. The method comprises the following steps: synthesizing sea urchin gold nanoparticles SUL-Au; then, performing surface modification on PEG by using the reaction of SUL-Au and methoxy polyethylene glycol sulfydryl; then carrying out surface modification on the surface of the beta-amyloid polypeptide to obtain an autophagy inhibitor Beclin 1; and finally, modifying SH-AS1411 to obtain SUL-Au @ PEG @ Beclin1@ AS 1411. The SUL-Au @ PEG @ Beclin1@ AS1411 prepared by the invention can target tumor cells to achieve the aim of directionally killing the tumor cells, and combines cell autophagy and photothermal therapy, thereby greatly improving the application efficiency of the gold nano material in tumor photothermal therapy.

Description

Synthesis method of gold nano-drug for targeted killing of tumor cells through cooperation of autophagy inhibition and photothermal therapy
Technical Field
The invention relates to the technical field of drug synthesis, in particular to a gold nano-drug synthesis method for inhibiting autophagy and killing tumor cells in a targeted manner by cooperation with photothermal therapy.
Background
Cancer, also known as malignant tumor, has become one of the major diseases threatening human life and health in the 2l century. The common cancer treatment methods at present include surgical treatment, chemical drug therapy and radiation therapy. The surgical treatment mainly refers to surgical removal of cancer tumor mass and tissues and organs affected by cancer tumor infiltration. Surgical treatment is effective for most malignant tumors and the most common treatment, but for advanced tumors and some tumors in sensitive areas, the risk of surgery is extremely high, the trauma to the patient is large, and a series of surgical complications are caused. Chemotherapy is the use of some chemicals that can damage the growth of cancer cells to prevent their growth. However, normal cell growth is also damaged, and some normal tissues and organs of the body have obvious toxic reactions. Radiotherapy is treatment using radiation generated by a radioisotope. The radiation can kill cancer tissue cells directly or indirectly, but the radiation inevitably affects and damages normal tissues of the human body, such as nausea, vomiting, leukopenia, inappetence and the like.
Photothermal therapy refers to that a photothermal agent having absorption in the near infrared absorbs the near infrared light and converts it into heat energy to kill malignant tumor cells. Malignant tumor tissue grows faster than normal tissue, and the blood vessels of the tumor tissue grow disorganized and malformed. Therefore, the capillary vessels in the tumor are easily pressed, the blood flow is not as large as that of normal tissues, most of the blood vessels of the tumor tissues are new blood vessels with low heat sensitivity, the heat resistance is not as good as that of the blood vessels of the normal tissues, the flow rate of the blood is slow, and the heat dissipation is slow, so that the temperature of the tumor tissue part rises very fast in the photothermal treatment process and is much higher than that of the surrounding normal tissues, and the temperature difference can reach as high as 10 ℃. When the temperature of the surrounding normal tissues reaches about 40 ℃ in the treatment process, the temperature of the tumor tissues can reach about 50 ℃, and the temperature difference can ensure that the normal tissues cannot be damaged when the temperature of the tumor tissues reaches the treatment critical temperature. And the high temperature environment can lead the cells to be in the environment with insufficient oxygen supply and low pH value, which further promotes the reduction of the high temperature resistance of the tumor tissue and further accelerates the death of the tumor tissue cells. Therefore, photothermal therapy has become an effective treatment for malignant tumors.
The key to photothermal therapy is to find a suitable photothermal agent. Good photothermal reagents need to have high thermal conversion efficiency and good biocompatibility. The nano gold material is always a hot point of research, and the surface plasma resonance peak (SPR peak) of the gold nanoparticles is related to factors such as the shape and the size of the nanoparticles, so that the position of the SPR peak can be regulated and controlled by controlling the morphology and the size of the nano gold, and the gold nano material also has the advantages of large light absorption cross section area, high photo-thermal conversion efficiency, good biocompatibility and the like. Hemicentrotus Seu Strongylocentrotus gold has been widely used in biological detection, cell labeling, and disease diagnosis due to its special structure. However, the traditional echinoid gold is synthesized by a template method, the synthesized size is uncontrollable, the synthesis efficiency is low, and the scientific research requirements cannot be well met, so that a simple, green and efficient synthesis method can be found out, which is the key for researching and developing anticancer drugs.
For example, patent CN107308462A discloses a green preparation method of echinoid nanogold and its application in tumor imaging and treatment, the invention mainly uses chloroauric acid as gold source, and sequentially adds stabilizer, reducing agent, growth inhibitor, and adopts a one-step synthesis method to prepare the echinoid nanogold. The method is simple, mild, green and environment-friendly. The synthesized echinoid nanogold can be used as a contrast agent to be applied to various medical imaging modes such as photoacoustic imaging, positron emission computed tomography, infrared thermal imaging and the like, a patient only needs to bear administration of one contrast agent, various diagnosis effects can be achieved, the dosage of the contrast agent can be reduced, and toxic and side effects are further reduced. In addition, the material can realize the conversion of light and heat under the irradiation of near infrared light, can absorb a large amount of rays, has higher photothermal conversion efficiency, and can be used for preparing a therapeutic agent for photothermal therapy, radiotherapy or combination therapy of tumor cells. However, the gold nanoparticles have complex synthesis steps, do not utilize the characteristics of the gold nanoparticles to effectively modify and synthesize the multifunctional nano material, and do not verify the therapeutic effect and the in vitro cell therapeutic effect, so that the true photothermal effect of the material cannot be verified.
Autophagy is an evolutionarily conserved important process in eukaryotes for turnover of intracellular material. In the process, some damaged proteins or organelles are wrapped by autophagy vesicles with double-layer membrane structures, and then are delivered into lysosomes (animals) or vacuoles (yeasts and plants) for degradation and recycling. Regulating autophagy, inhibiting tumor growth, and enhancing killing effect of chemotherapy drugs and nanometer anticancer agent on cancer cells.
For example, patent CN104353074A discloses a method for killing tumor cells by combining gold-mediated near infrared thermal effect and autophagy inhibitor, compared with the prior art, the introduction of autophagy inhibitor in the invention can reduce the dosage of gold nanoparticles, and a good tumor cell killing effect can be obtained by using a lower dosage of laser, thereby reducing the risk of harm of laser and gold nanoparticles to normal cell tissues. However, all the gold nanoparticles are gold nanocages, so that the photo-thermal conversion efficiency is low, the specific surface area is small, and the decorative objects are few; and has no targeting property and no targeted treatment effect on tumors.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a synthesis method of a gold nano-drug for inhibiting autophagy and killing tumor cells in a targeted manner by cooperating photothermal therapy; the invention utilizes self-synthesized echinoid gold nanoparticles (SUL-Au) AS a platform, and PEG, Beclin1 and AS1411 are modified on the surface of the platform to synthesize a novel nano-drug SUL-Au @ PEG @ Beclin1@ AS1411, which can target tumor cells and realize the purpose of directionally killing the tumor cells, and combines cell autophagy and photothermal therapy, thereby greatly improving the application efficiency of the gold nano-material in tumor photothermal therapy.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a synthesis method of a gold nano-drug for inhibiting autophagy and treating tumor cells in a targeted manner by cooperation with photothermal therapy, which comprises the following steps:
step 1: synthesis of SUL-Au
30mL of water was added to a round bottom flask, and an amount of 300mM HAuCl was added4Heating the aqueous solution with stirring, adding sodium citrate aqueous solution, sodium citrate and HAuCl when the solution is boiling4Mixing according to the mass ratio of 1:3, stirring and heating for 10min under the condition of keeping boiling until the solution turns to wine red, stopping heating, cooling to room temperature, and completing the synthesis of gold seeds Au.
Adding proper amount of water into a reaction bottle, and adding a certain amount of HAuCl4Stirring the aqueous solution, adding the gold seed solution according to the volume ratio of 1:100, adding a proper amount of 30mM hydroquinone solution, stirring for 40min at room temperature until the solution becomes bluish purple, stopping stirring, synthesizing echinoid gold nanoparticles, wherein the echinoid gold has the size of about 100nm and obvious convex surfaces.
Step 2: synthesis of SUL-Au @ PEG
1mL of 100ug/mLAu solution is taken, added with 1.5mL of SH-m PEG 1000(20ug/mL) and reacted for 12h at room temperature;
and centrifuging the product at 8500r/min for 10min, removing the supernatant, and adding 1mL of deionized water to prepare SUL-Au @ PEG.
And step 3: synthesis of SUL-Au @ PEG @ Beclin1
0.01mL of Beclin 1(250ug/mL) was added to 1mL of SUL-Au @ PEG solution (100ug/mL) and reacted at room temperature for 24 h;
the product was centrifuged at 8500r/min for 10min, the supernatant removed and 1mL of deionized water added.
And 4, step 4: synthesis of SUL-Au @ PEG @ Beclin1@ AS1411
0.01mL of SH-AS1411(10uM/mL) was added to 1mL of SUL-Au @ PEG @ Beclin1 solution (100ug/mL) and reacted at room temperature for 12 h;
the product was centrifuged at 8500r/min for 10min, the supernatant removed and 1mL of deionized water added.
Compared with the prior art, the invention has the following beneficial effects:
the SUL-Au @ PEG @ Beclin1@ AS1411 nano-particle has the advantages of simple and green synthesis steps, easy operation, capability of meeting the requirement of environment-friendly social development, uniform size of the synthesized nano-particle, good dispersion effect, appearance similar to that of sea urchin, high photo-thermal conversion efficiency and high decoration property. Under the irradiation of infrared laser with wavelength of 808nm, the SUL-Au @ PEG @ Beclin1@ AS1411 can obviously improve the targeting property and the high efficiency of the echinoid gold nanoparticles in the aspect of tumor photothermal treatment, and has a very wide application prospect in the aspect of clinical tumor treatment.
Drawings
FIG. 1 is a TEM representation of SUL-Au synthesized in example 1 of the present invention;
FIG. 2 is a UV absorption spectrum of SUL-Au @ PEG @ Beclin1@ AS 1411;
FIG. 3 is a photo-thermal efficiency picture of SUL-Au @ PEG @ Beclin1@ AS 1411;
FIG. 4 is a graph showing cytotoxicity tests of SUL-Au @ PEG @ Beclin1@ AS 1411.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description will be made with reference to specific drawings and embodiments.
In the present invention, the materials and reagents used are not specifically described, and are commercially available. PEG was purchased from Shanghai Peng Biotech, BECLIN1 was purchased from Gill Biochemical (Shanghai), AS1411 was purchased from Bioengineer (Shanghai).
The invention provides a synthesis method of a gold nano-drug for inhibiting autophagy and killing tumor cells in a targeted manner by cooperating with photothermal therapy, and the specific embodiment is as follows.
Example 1
A synthesis method of a gold nano-drug for targeted killing of tumor cells by autophagy inhibition and photothermal therapy comprises the following steps:
step 1: synthesis of SUL-Au
30mL of water was added to a round-bottomed flask, and 25uL of 300mM HAuCl was added4Heating the aqueous solution with stirring, adding 900Ul of 1 w/v% aqueous sodium citrate, sodium citrate and HAuCl when the solution is boiling4Heating under stirring for 10min until the solution turns wine red, stopping heating, cooling to room temperature, and collecting goldSeed Au synthesis was complete.
In a reaction flask, 10mL of deionized water was added, and 10uL of HAuCl was added at a concentration of 300mM4Stirring the aqueous solution, adding the gold seed solution according to the volume ratio of 1:100, adding 1mL of 30mM hydroquinone solution, stirring for 40min at room temperature until the solution becomes bluish purple, stopping stirring, synthesizing echinus gold nanoparticles, and adding water to adjust the concentration to 100 ug/mL; the characterization measurement is carried out by using a transmission electron microscope, the size of the sea urchin-like gold is about 100nm, and the surface has obvious convex shapes, as shown in figure 1.
And 2, step: synthesis of SUL-Au @ PEG
Taking 1mL of 100ug/mL SUL-Au solution, adding 1.5mL of SH-m PEG 1000(20ug/mL), and reacting at room temperature for 12 h;
centrifuging the product at 8500r/min for 10min, removing supernatant, and adding deionized water to obtain 100ug/mL SUL-Au @ PEG solution;
and step 3: synthesis of SUL-Au @ PEG @ Beclin1
0.01mL of Beclin 1(250ug/mL) was added to 1mL of SUL-Au @ PEG solution (100ug/mL) and reacted at room temperature for 24 h;
centrifuging the product at 8500r/min for 10min, removing the supernatant, and adding deionized water to obtain 100ug/mL SUL-Au @ PEG @ Beclin1 solution;
and 4, step 4: synthesis of SUL-Au @ PEG @ Beclin1@ AS1411
0.01mL of SH-AS1411(10uM/mL) was added to 1mL of SUL-Au @ PEG @ Beclin1 solution (100ug/mL) and reacted at room temperature for 12 h;
and centrifuging the product at the rotating speed of 8500r/min for 10min, removing the supernatant, and adding 1mL of deionized water to obtain a SUL-Au @ PEG @ Beclin1@ AS1411 solution.
The prepared SUL-Au @ PEG @ Beclin1@ AS1411 was subjected to the relevant performance test AS follows.
FIG. 1 is a TEM transmission electron micrograph of SUL-Au, which shows that the synthesized SUL-Au nanoparticles have uniform size, particle size of about 100nm, uniform dispersion, simple synthesis method and easy application.
1. Ultraviolet characterization SUL-Au @ PEG @ Beclin1@ AS1411
Ultraviolet absorption spectrum detection was performed on the SUL-Au solution prepared in step 1 and the SUL-Au @ PEG @ Beclin1@ AS1411 prepared in example 1 of the present invention, AS shown in fig. 2.
As is clear from FIG. 2, the absorption peak of the SUL-Au solution is around 608nm, and after PEG, Beclin1 and AS1411 were sequentially modified on the surface thereof, the absorption peak of SUL-Au @ PEG @ Beclin1@ AS1411 was 660nm, and the absorption peak was red-shifted. The SUL-Au @ PEG @ Beclin1@ AS1411 is proved to be capable of improving the near infrared light absorption effect and improving the photo-thermal conversion efficiency. Namely, the SUL-Au @ PEG @ Beclin1@ AS1411 prepared by the invention has better infrared absorption for 808nm infrared light irradiation, thereby realizing higher photo-thermal conversion efficiency, and is shown in figure 2.
2. Photothermal efficiency of SUL-Au @ PEG @ Beclin1@ AS1411
The power density is 2.0W/cm2In this example, a PBS solution of a blank control, a SUL-Au @ PEG @ Beclin1@ AS1411 solution (50ug/mL) (solvent: PBS) was irradiated with near infrared NIR having a wavelength of 808nm, and the temperature profile with the irradiation time was recorded, AS shown in FIG. 3.
As can be seen from FIG. 3, the temperature of SUL-Au @ PEG @ Beclin1@ AS1411 rises to about 79 ℃ after NIR irradiation for 10 min; while the temperature of the PBS solution remained substantially unchanged from the original temperature. The SUL-Au @ PEG @ Beclin1@ AS1411 is shown to be capable of rapidly absorbing near infrared light and efficiently converting into heat energy. The SUL-Au @ PEG @ Beclin1@ AS1411 prepared by the method has high photo-thermal conversion efficiency, and can convert absorbed near infrared light into heat.
3. Cytotoxicity assays for SUL-Au @ PEG @ Beclin1@ AS1411
MCF-7 cells (breast cancer cells) were added to 96-well plates at 1X 10 per well4Culturing the cells in an incubator for 18h, sucking out the culture solution when the cells adhere to the wall and the single hole is full of about 70% -80%, then adding DMEM cell culture solution respectively containing SUL-Au @ PEG, SUL-Au @ PEG @ Beclin1@ AS1411 with different final concentrations into a 96-well plate, wherein the concentrations are 50ug/mL, 40ug/mL, 30ug/mL, 20ug/mL and 10ug/mL in sequence, and after incubating for 8h, using 2.0W/cm2NIR irradiation with 808nm infrared light for 2 minutes, incubation for 1 hour and aspiration of the culture medium. Adding 10uL of CCK8 and 90uL of culture solution into each well, and standing for cultureIncubating for 30min, and placing into enzyme labeling instrument to detect cell activity, with the result shown in FIG. 4.
As can be seen from FIG. 4, the SUL-Au @ PEG and the SUL-Au @ PEG @ Beclin1@ AS1411 synthesized by the method have no toxicity to cells, but after adding NIR light of 808nm, the SUL-Au @ PEG @ Beclin1@ AS1411 has a remarkable killing effect on MCF-7 cells compared with the SUL-Au @ PEG of unmodified autophagy inhibitors Beclin1 and AS1411, and the synthesized material has a good photothermal treatment effect.
In conclusion, the invention synthesizes a novel nano-drug SUL-Au @ PEG @ Beclin1@ AS1411 by using self-synthesized echinoid gold nanoparticles (SUL-Au) AS a platform, which can target tumor cells to achieve the purpose of directionally killing the tumor cells, and combines cell autophagy and photothermal therapy, thereby greatly improving the application efficiency of the gold nano-material in tumor photothermal therapy.
The foregoing is a preferred embodiment of the present invention, and it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and are intended to be within the scope of the invention.

Claims (7)

1. A synthesis method of a gold nano-drug for inhibiting autophagy and killing tumor cells in a targeted manner by using photothermal therapy is characterized by comprising the following steps:
step 1: synthesizing sea urchin gold nanoparticles SUL-Au;
step 2: synthesizing SUL-Au @ PEG;
reacting the SUL-Au prepared in the step 1 with methoxypolyethylene glycol sulfydryl at room temperature, centrifuging a product, and removing a supernatant to obtain SUL-Au @ PEG;
and step 3: synthesizing SUL-Au @ PEG @ Beclin 1;
adding an autophagy inhibitor Beclin1 into the SUL-Au @ PEG solution prepared in the step 2, reacting at room temperature, centrifuging the product, and removing the supernatant to obtain SUL-Au @ PEG @ Beclin 1;
and 4, step 4: synthesizing SUL-Au @ PEG @ Beclin1@ AS 1411;
and (3) adding an SH-AS1411 aptamer into the SUL-Au @ PEG @ Beclin1 solution prepared in the step (3), reacting at room temperature, centrifuging the product, and removing the supernatant to obtain the SUL-Au @ PEG @ Beclin1@ AS 1411.
2. The method for synthesizing gold nano-drugs for targeted killing of tumor cells by autophagy inhibition and photothermal therapy according to claim 1, wherein the step 1 comprises synthesis of gold seed solution and synthesis of echinocandin nanoparticles.
3. The method for synthesizing gold nano-drugs for inhibiting autophagy and treating targeted tumor cell killing by cooperation with photothermal therapy according to claim 2, wherein the synthesis of the gold seed solution specifically comprises the following steps: 30mL of water was added to a round-bottomed flask, and a certain amount of 300mM HAuCl was added4Heating the aqueous solution with stirring, adding sodium citrate aqueous solution when the solution is boiling, wherein the sodium citrate is mixed with HAuCl4Mixing according to the mass ratio of 1: 3; heating under stirring for 10min until the solution turns wine red, stopping heating, and cooling to room temperature to obtain gold seed solution.
4. The method for synthesizing gold nano-drugs for inhibiting autophagy, cooperating with photothermal therapy, and targeted killing of tumor cells according to claim 3, wherein the synthesis of echinocandin nano-particles specifically comprises: adding a proper amount of water into a reaction bottle, and adding a certain amount of HAuCl4And (3) stirring the aqueous solution, then adding the gold seed solution according to the volume ratio of 1:100, adding a proper amount of 30mM hydroquinone solution, stirring for 40min at room temperature until the solution becomes bluish purple, and stopping stirring to obtain the echinacea gold nanoparticles.
5. The method for synthesizing gold nano-drugs for inhibiting autophagy and treating targeted tumor cell killing by cooperation with photothermal therapy according to claim 1, wherein the step 2 specifically comprises the following steps: taking 1mL of 100ug/mL SUL-Au solution prepared in the step 1, adding 1.5mL of 20ug/mL methoxy polyethylene glycol mercapto (SH-mPEG 1000), and reacting at room temperature for 12 h; centrifuging the product at 8500r/min for 10min, removing supernatant, and adding deionized water to obtain 100ug/mL SUL-Au @ PEG.
6. The synthesis method of the gold nano-drug for inhibiting autophagy and killing tumor cells in a targeted manner by using photothermal therapy in cooperation with the cooperation of the autophagy inhibition according to claim 1, wherein the step 3 is specifically as follows: adding 0.01mL of 250ug/mL autophagy inhibitor Beclin1 into 1mL of SUL-Au @ PEG solution, and reacting at room temperature for 24 h; the product was centrifuged at 8500r/min for 10min, the supernatant removed and deionized water added to produce 100ug/mL SUL-Au @ PEG @ Beclin 1.
7. The synthesis method of the gold nano-drug for inhibiting autophagy and killing tumor cells in a targeted manner by using photothermal therapy in cooperation with the cooperation of the autophagy inhibition according to claim 1, wherein the step 4 is specifically as follows: adding 0.01mL of 10uM/mL SH-AS1411 aptamer into 1mL of SUL-Au @ PEG @ Beclin1 solution, and reacting for 12h at room temperature; and centrifuging the product at the rotating speed of 8500r/min for 10min, removing the supernatant, and adding 1mL of deionized water to obtain SUL-Au @ PEG @ Beclin1@ AS 1411.
CN202210103500.2A 2022-01-27 2022-01-27 Synthesis method of gold nano-drug for targeted killing of tumor cells through cooperation of autophagy inhibition and photothermal therapy Pending CN114617963A (en)

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CN102328093A (en) * 2011-08-30 2012-01-25 吉林大学 Method for preparing gold nano particles with echinoid structures by seed intermediate approach
CN104353074A (en) * 2014-10-15 2015-02-18 中国科学技术大学 Method for killing tumor cells through combination of gold-mediated near-infrared light heat effect and autophagy inhibitor

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Application publication date: 20220614