CN111184862B - Preparation method and application of composite nano gold particles - Google Patents
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Abstract
The invention belongs to the technical field of nano material preparation, and particularly relates to a preparation method and application of composite nano gold particles. The method comprises the steps of reducing a gold solution by using sodium borohydride to obtain a gold seed solution, adding the gold seed solution and a gold ion solution into a hyaluronic acid solution, heating to grow gold seeds, and adding indocyanine green and chitosan solutions respectively to obtain stable composite nano gold particles. The invention has the characteristics of green color, simple method and mild reaction conditions, and the prepared nanoparticles can be used for photoacoustic synergistic treatment of tumors.
Description
Technical Field
The invention belongs to the technical field of nano material preparation, and particularly relates to a preparation method and application of composite nano gold particles.
Background
Nanomaterials play an important role in the development of nanoscience and nanotechnology, and nanostructures exhibit different physical and chemical properties compared to small molecules or small volume materials. Among the different types of nanomaterials, metal nanoparticles, in particular nanogold particles, have attracted considerable interest to researchers in different fields, mainly due to the unique properties of nanogold (high X-ray absorption coefficient, ease of synthesis, precise control of the physicochemical properties of the particles, unique tunable optics and different electronic properties).
Gold is one of the earliest metals discovered by humans and has a long history of research and application. The first material on colloidal gold was found by scientists in china, arabian and india in ancient books, who obtained colloidal gold as early as the 5 and 4 th century before the c.yuan and applied it to medical applications. In 1857, Faraday made systematic scientific research on nanogold, and his discovery laid a scientific foundation for the practical application of nanogold particles.
The application of nanotechnology to the field of biology has resulted in nanotechnology, the development of which has had a profound impact on medicine. Some drugs based on nanotechnology have appeared on the market over the last decades, and many nanomaterials with drug diagnosis and drug delivery functions are applied in biomedicine. The nano gold particles have the characteristics of unique optical property, good biocompatibility, easiness in modification of biomolecules, simplicity in preparation and the like, so that the nano gold particles are widely researched in the biomedical fields of biosensing, molecular imaging, tumor treatment, drug delivery and the like.
At the beginning of the development of nano-technology, a number of technologists are exploring the preparation technology of nano-gold particles. To date, various methods for preparing gold nanoparticles have been discovered, which can be generally classified into physical methods, chemical methods, and green biosynthesis methods. Methods of gamma-irradiation, microwave irradiation, sonochemical, ultraviolet irradiation, laser ablation, pyrolytic processes, and photochemical processes are generally considered physical methods. The chemical method is to take a gold compound as a raw material, generate gold nanoparticles by a reduction reaction, and prepare particles with required sizes by controlling reaction conditions. The chemical method mainly comprises the following steps: water phase oxidation-reduction method, seed crystal method, microemulsion method, template method, etc. With the popularization of green chemistry concept, the synthesis of nano-gold is also developed towards the direction of no toxicity and environmental friendliness. Generally, the green synthesis of nanogold is carried out by adopting biological materials such as microorganisms and plants.
The method adopts a seed-mediated growth method (seed crystal method) to synthesize the nanogold, small-particle gold is used as a seed, gold solution in an ionic state is added into a system, and a proper amount of reducing agent is added to enable the surface of the nanogold particle to generate autocatalytic reaction, so that the seed crystal grows gradually. The method belongs to the chemical synthesis of nano-gold, and related researchers think that the chemical method is still the first choice for preparing nano-gold.
Photothermal therapy (PTT), known as thermal ablation or optical hyperthermia, is a minimally invasive tumor therapy and one of the most promising approaches to the treatment of cancer and infectious diseases today. Nanogold absorbs the most in the visible or near infrared region and becomes very hot under corresponding light irradiation. If in this case they are located in or around the target cells, these cells will die by binding the gold particles to the antibodies or other molecules.
Substances in photodynamic therapy (PDT) can selectively accumulate in tumors or other target tissues (cells). The affected tissue is irradiated with laser light at a wavelength corresponding to the dye absorption peak. In this case, in addition to the heat generated by absorption. Another mechanism plays a crucial role, namely the generation of photochemical singlet oxygen and the formation of highly reactive free radicals, which can induce tumor cell necrosis and apoptosis.
The sonodynamic therapy (SDT) utilizes ultrasonic waves to penetrate biological tissues, particularly focused ultrasonic waves to focus sound energy on deep tissues in an atraumatic manner, and activates some sound-sensitive drugs to generate an anti-tumor effect.
Indocyanine green (ICG) is a water-soluble dye with strong absorption in the near infrared region, and is a few dyes approved for clinical use by the FDA in the united states. The near-infrared fluorescence/photoacoustic bimodal imaging system can be used for near-infrared fluorescence/photoacoustic bimodal imaging and photoacoustic power/photothermal synergistic treatment. ICG stability, however, is poor, including water instability, thermal degradation, and photodegradation.
Chitosan is the only cationic polymer with positive charge discovered at present, has good biocompatibility, and has been widely used as a drug delivery carrier. Sodium hyaluronate is a dextran aldehyde acid, an inherent component in the human body. Through the interaction of positive and negative charges, the chitosan can react with hyaluronic acid to form polyelectrolyte composite nano-particles.
Therefore, the ICG stability can be improved by preparing the composite nano-gold particles and utilizing the composite nano-particles formed by chitosan and hyaluronic acid, and the ICG stability and the nano-gold particles generate a synergistic effect to improve the killing effect of the ICG on tumor cells.
Disclosure of Invention
The invention aims to provide a preparation method and application of composite nano gold particles; the composite nano gold particles are obtained by the attraction of positive and negative charges of chitosan and hyaluronic acid and the combination with the nano gold particles. The prepared composite nano gold particles can be used for photoacoustic synergistic treatment of tumors.
In order to achieve the purpose, the invention also adopts the following technical scheme:
a preparation method of composite nano gold particles comprises the following steps:
step S1, reducing the gold solution by using sodium borohydride to obtain a gold seed solution, adding the gold seed solution and the gold ion solution into the hyaluronic acid solution, and heating to grow the gold seeds to obtain nano gold particles;
step S2, adding an indocyanine green solution into a nano gold particle solution, and incubating the indocyanine green solution and the nano gold particle solution at room temperature; then slowly adding the chitosan solution at the dropping speed of 1 drop/s to form nano particles; and (5) incubating again to prepare the stable composite nano gold particles.
Further, the step S1 specifically includes the following steps:
step S11, adding the sodium borohydride solution into the chloroauric acid solution, and placing the chloroauric acid solution on a magnetic stirrer to stir and react to obtain a gold seed solution;
and step S12, adding the gold seed solution into the mixed solution of the hyaluronic acid solution and the chloroauric acid solution, and heating to obtain the gold nanoparticles.
Further, the chloroauric acid solution used in step S11, wherein Au is3+The concentration of (A) is 0.1 to 1 mM; the concentration of the sodium borohydride solution is 0.05-0.15M.
Further, in step S11, the stirring reaction is: the reaction temperature is 25-35 ℃, and the reaction time is 2-4 h.
Further, the chloroauric acid solution used in step S12, wherein Au is3+The concentration of (A) is 1 to 5 mM; the concentration of the hyaluronic acid solution is 0.5-2 mg/mL.
Further, in step S12, the heating specifically includes: the reaction temperature is 95-105 ℃, and the reaction time is 5-15 min.
Further, the incubation in the step S2 is specifically incubation for 20min to 1h at room temperature; and the secondary incubation time is 24-72 h.
Further, the concentration of the indocyanine green in the step S2 is 0.05-0.5 mu g/mL; the concentration of the chitosan is 0.5-2 mg/mL, and the solvent is an acetic acid solution with the volume ratio of 1%; the volume ratio of the nano gold particle solution to the chitosan solution is 3: 1-4: 1.
The invention also aims to provide the composite nano gold particles prepared by the preparation method.
The invention also aims to provide the application of the composite nano gold particles prepared by the preparation method in preparing tumor treatment medicines.
The invention has the following remarkable advantages:
according to the invention, the nano gold particles are prepared from sodium borohydride and hyaluronic acid, so that the nano gold particles suspended in hyaluronic acid can be synthesized in a green manner simply, indocyanine green solution is added into the solution, and after incubation for a period of time, chitosan solution is added to form composite nano gold particles, and the composite nano gold particles are applied to tumor treatment.
Drawings
FIG. 1 is a particle size diagram of the composite gold nanoparticles of the present invention.
FIG. 2 is a transmission electron microscope image of the composite nano-gold particles of the present invention.
FIG. 3 is a UV scan of the composite gold nanoparticles of the present invention.
FIG. 4 is a graph showing the stability test results of the composite gold nanoparticles of the present invention.
FIG. 5 is a diagram showing the killing effect of the composite gold nanoparticles of the present invention on tumor cells.
Detailed Description
For further disclosure, but not limitation, the present invention is described in further detail below with reference to examples.
Example 1
A preparation method of gold nano-gold particles comprises the following steps:
(1) preparing a synthetic reaction solution: the preparation concentration of Au is 0.25mmol/L3+Solution (chloroauric acid, HAuCl)4·3H2O) 20mL was placed in a 50mL flask. And preparing a gold solution of 3mmol/L for later use. Sodium borohydride solution with the concentration of 0.1M is prepared and placed in an ice water bath. A1 mg/mL hyaluronic acid solution was prepared with ultrapure water and stirred overnight.
(2) Synthesizing a gold seed solution: 0.6mL of sodium borohydride solution (0.1M) was added to 20mL of gold solution (0.25 mmol/L) under magnetic stirring, and reacted at 25 ℃ for 3 h.
(3) And (3) synthesizing nano gold: the gold seed solution 700. mu.L from step (2) was added to 1mL of hyaluronic acid solution (1 mg/mL), and 100. mu.L of gold solution (3 mmol/L) was added and heated at 100 ℃ for 10 min.
Example 2
A preparation method of composite nano gold particles comprises the following steps:
(1) preparing a solution: firstly preparing 1% acetic acid solution, preparing 1mg/mL chitosan solution by using the acetic acid solution, putting the solution into a shaking table, standing overnight, and filtering by using a membrane.
(2) Synthesizing nano particles: and taking 700 mu L of the nanogold solution suspended in the hyaluronic acid in the example 1, adding 200 mu L of the chitosan solution, and slowly mixing to obtain the composite nanogold particles without ICG. Is named ACH.
Example 3
A preparation method of composite nano gold particles comprises the following steps:
(1) preparing a solution: firstly preparing 1% acetic acid solution, preparing 1mg/mL chitosan solution by using the acetic acid solution, putting the solution into a shaking table, standing overnight, and filtering by using a membrane. Indocyanine green was dispersed in ultrapure water at a concentration of 1 mg/mL.
(2) Synthesizing nano particles: taking 700 mu L of the nanogold solution suspended in hyaluronic acid in example 1, adding 10 mu L of indocyanine green solution, mixing uniformly, and incubating for 30 min. Adding 200 mu L of chitosan solution, slowly and uniformly mixing to obtain the ICG-coated composite nano gold particles. Is named ACHI.
Example 4
Firstly, 1% acetic acid solution is prepared, and then 1mg/mL chitosan solution is prepared by using the acetic acid solution. Slowly mixing the chitosan solution according to the volume ratio of 1:3.5 (chitosan: hyaluronic acid) (1 drop/s), incubating for 2h, centrifuging (5000 r/s, 1 min), and collecting the supernatant to obtain the composite nanoparticles. Designated as AC.
Example 5
ACHI prepared in example 2 was added with PBS to prepare a solution having a concentration of 80. mu.g/mL, and the solution was left at room temperature for 7 days, and samples were taken for 1, 3, 5, and 7 days, respectively, to measure the change in particle size.
ACHI prepared in example 2 was added to 1640 medium containing 10% fetal bovine serum to prepare a solution having a concentration of 80. mu.g/mL, the solution was left at room temperature for 7 days, and samples were taken for 1, 3, 5, and 7 days, respectively, to measure the change in particle size.
Application example 1
Evaluating the killing effect of the tumor cells, comprising the following steps:
(1) culturing the cells: pc9 cells (human lung carcinoma cells) were cultured in 1640 medium containing 10% FBS, 1% diabody (penicillin, streptomycin) and 5% CO2Culturing at 37 deg.C, spreading the cells in 96-well plate with density of 1 × 105。
(2) The killing effect of the composite nano gold particles prepared by the invention on cells is detected as follows: the nanoparticles were added to a 96-well plate at different concentrations and after 4h, sonicated (0.3W, 1 min) and irradiated with 808nm (2.5W, 3 min) laser, respectively.
(3) Evaluation: after 48h, 10% MTT was prepared with the medium, and the medium in the 96-well plate was aspirated and 100. mu.L of MTT was added. After 4h, the medium was aspirated, 150. mu.L of dimethyl sulfoxide was added, shaken well, absorbance was measured at 490nm with a microplate reader, and the cell viability was calculated.
Fig. 1 is a particle size distribution diagram of ACHI obtained in example 3, and the result shows that both indocyanine green and nanogold are encapsulated in composite nanoparticles formed by chitosan and hyaluronic acid, so as to form a uniform nanoparticle solution. The composite nano gold particles have uniform particle size distribution, and the average particle size is about 250 nm.
FIG. 2 is a transmission electron micrograph of ACHI obtained in example 3. It can be clearly seen from the figure that the nanogold particles are wrapped in the composite nanoparticle.
FIG. 3 is a comparison of the UV spectra of ACH obtained in example 2, ACHI obtained in example 3, and AC obtained in example 4. As can be seen, ICG has a strong absorption at about 778nm, while ACHI has no strong absorption peak at 778nm and a strong absorption peak at 698 nm. The results indicate that nanogold and indocyanine green are coated in a complex formed by chitosan and hyaluronic acid, forming composite nanogold particles.
FIG. 4 shows the particle size change of ACHI obtained in example 3 in PBS and 1640 medium containing serum. The result shows that the composite nano gold particles have good stability in PBS and 1640 culture medium containing serum, and can be used for subsequent biological research.
FIG. 5 shows the tumor cell inhibitory effect of ACHI obtained in example 3. The Control group was a Control group, i.e., after adding nanoparticles to the cells, no other treatment was performed. Ultrasound is a sonication group, i.e., cells are treated with Ultrasound after nanoparticles are added thereto. NIR is a laser irradiation treatment group, i.e.after addition of nanoparticles on the cells, treatment with 808nm laser irradiation. The Ultrasound + NIR optical/acoustic combination treatment group, i.e.after the addition of nanoparticles on the cells, was treated with 808nm laser irradiation followed by sonication. The results show that the composite nano gold particles have good light/sound cancer inhibition effect. When photoacoustic synergy is used to kill cancer cells, the effect is better than using light/sound treatment alone.
Claims (10)
1. A preparation method of composite nano gold particles is characterized by comprising the following steps: the method comprises the following steps:
step S1, reducing the gold solution by using sodium borohydride to obtain a gold seed solution, adding the gold seed solution and the gold ion solution into the hyaluronic acid solution, and heating to grow the gold seeds to obtain nano gold particles;
step S2, adding an indocyanine green solution into a nano gold particle solution, and incubating the indocyanine green solution and the nano gold particle solution at room temperature; then slowly adding the chitosan solution at the dropping speed of 1 drop/s to form nano particles; and (5) incubating again to prepare the stable composite nano gold particles.
2. The method for preparing composite nano gold particles according to claim 1, wherein the method comprises the following steps: the step S1 specifically includes the following steps:
step S11, adding the sodium borohydride solution into the chloroauric acid solution, and placing the chloroauric acid solution on a magnetic stirrer to stir and react to obtain a gold seed solution;
and step S12, adding the gold seed solution into the mixed solution of the hyaluronic acid solution and the chloroauric acid solution, and heating to obtain the gold nanoparticles.
3. The method for preparing composite nano gold particles according to claim 2, wherein the method comprises the following steps: chloroauric acid solution used in step S11, wherein Au3+The concentration of (A) is 0.1 to 1 mM; the concentration of the sodium borohydride solution is 0.05-0.15M.
4. The method for preparing composite nano gold particles according to claim 2, wherein the method comprises the following steps: the stirring reaction in the step S11 is as follows: the reaction temperature is 25-35 ℃, and the reaction time is 2-4 h.
5. The method for preparing composite nano gold particles according to claim 2, wherein the method comprises the following steps: chloroauric acid solution used in step S12, wherein Au3+Is rich inThe degree is 1-5 mM; the concentration of the hyaluronic acid solution is 0.5-2 mg/mL.
6. The method for preparing composite nano gold particles according to claim 2, wherein the method comprises the following steps: the heating in step S12 is specifically: the reaction temperature is 95-105 ℃, and the reaction time is 5-15 min.
7. The method for preparing composite nano gold particles according to claim 1, wherein the method comprises the following steps: the incubation at the room temperature in the step S2 is specifically incubation for 20 min-1 h; and the secondary incubation time is 24-72 h.
8. The method for preparing composite nano gold particles according to claim 1, wherein the method comprises the following steps: the concentration of the indocyanine green in the step S2 is 0.05-0.5 mu g/mL; the concentration of the chitosan is 0.5-2 mg/mL, and the solvent is an acetic acid solution with the volume ratio of 1%; the volume ratio of the nano gold particle solution to the chitosan solution is 3: 1-4: 1.
9. The composite nano gold particles prepared by the preparation method according to any one of claims 1 to 8.
10. The use of the composite gold nanoparticles prepared by the preparation method according to any one of claims 1 to 8 in the preparation of a medicament for treating tumors.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106832059A (en) * | 2017-03-08 | 2017-06-13 | 福州大学 | A kind of Tarceva Cy7 chitosan polymers with tumor-targeting |
| CN108210925A (en) * | 2018-04-03 | 2018-06-29 | 国家纳米科学中心 | A kind of Nano medication and its preparation method and application |
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| CN106832059A (en) * | 2017-03-08 | 2017-06-13 | 福州大学 | A kind of Tarceva Cy7 chitosan polymers with tumor-targeting |
| CN108210925A (en) * | 2018-04-03 | 2018-06-29 | 国家纳米科学中心 | A kind of Nano medication and its preparation method and application |
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| Title |
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| "Biopolymer coated gold nanocrystals prepared using the green chemistry approach and their shape-dependent catalytic and surface-enhanced Raman scattering properties";Chih-Wei Chou等;《Phys. Chem. Chem. Phys.》;20130507(第15期);第11275-11286页 * |
| "Rapid Synthesis and Catalytic Application of Biocompatible HA-Au Nanoparticles";Chih-Wei Chou等;《Proceedings of the 2011 6th IEEE International》;20111231;第1008-1011页 * |
| "透明质酸修饰的介孔二氧化硅包覆金纳米棒的制备及在肿瘤化疗-热疗联合治疗中的应用";金新天等;《高等学校化学学报》;20160229;第37卷(第2期);第224-231页 * |
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