CN109396457A

CN109396457A - A kind of shape and the controllable nanogold particle and the preparation method and application thereof of size

Info

Publication number: CN109396457A
Application number: CN201811482468.3A
Authority: CN
Inventors: 肖玉玲; 洪学传; 吴华萍; 唐琳; 曾小东
Original assignee: SUZHOU INSTITUTE OF WUHAN UNIVERSITY
Current assignee: SUZHOU INSTITUTE OF WUHAN UNIVERSITY
Priority date: 2018-12-05
Filing date: 2018-12-05
Publication date: 2019-03-01
Anticipated expiration: 2038-12-05
Also published as: CN109396457B

Abstract

The invention discloses the controllable nanogold particles and the preparation method and application thereof of a kind of shape and size.Using seed mediated growth method synthesizing water-solubility, it is monodispersed, the gold nano grain of different-shape size, including spherical shape, it is rodlike, dumbbell shaped, bipyramid shape, cubic and branched structure it is starlike etc., the serial nano gold is with ascorbic acid, sodium borohydride or hydroquinone are as reducing agent, cetyl trimethylammonium bromide is as cationic surfactant and stabilizer, sodium citrate is as reducing agent and stabilizer, it can get various sizes of nanogold by changing seed solution, realize the finely regulating of nanogold pattern and partial size, high yield, high-purity, good aqueous solubility, no biotoxicity, purifying process is simple.It can be applied to the fields such as photoacoustic imaging, photo-thermal therapy, bio-sensing.

Description

Shape and size controllable gold nanoparticle and preparation method and application thereof

Technical Field

The invention belongs to the technical field of nano material preparation, relates to tumor imaging, photothermal therapy and the like in the field of biomedicine, and particularly relates to a preparation method of a series of nano gold particles with different shapes and sizes.

Background

Cancer has become a leading enemy of human health. Conventional treatment modalities include chemotherapy, radiation therapy, surgical resection, and the like. However, due to the challenges of poor patient compliance during chemotherapy, high surgical recurrence probability, damage to normal cells by radiotherapy, and the like, novel and effective cancer treatment strategies need to be researched. Targeted therapy is an emerging class of therapies that has evolved gradually from the late 90 s of the 20 th century and has achieved significant effects in the treatment of certain types of cancer. The nanometer material is used as a drug or gene transmission carrier, has the advantages of improving the water solubility and stability of the drug, reducing the toxicity of the drug, enhancing the curative effect of the drug, improving the distribution of the drug in vivo and the like, and develops rapidly in recent years.

Today, nano-gold shows huge potential in the biomedical field by virtue of unique physicochemical property and optical property thereof, and research of nano-gold also gets very extensive attention. The nano gold is a gold material with the grain diameter of 1nm-250nm, is easy to synthesize, has small toxicity and good biocompatibility, can accurately regulate and control the optical property of the nano gold, and is easy to be used for modifying medicines and targeting molecules. As nano metal particles, surface electrons can only vibrate in a nano size, thus generating a local plasma phenomenon, and when near infrared light with a proper wavelength is used for irradiation, the metal particles can absorb light energy and convert the light energy into heat energy, namely, a photothermal effect. And the nano gold is used as inert metal, and in an excitation area, because of the enhancement of a near-surface electromagnetic field, a Raman scattering signal absorbed by molecules is greatly enhanced compared with that of common Raman scattering. By utilizing the Surface Enhanced Raman Scattering (SERS) and local plasmon resonance (LSPR) effects of the nanogold, the nanogold becomes a research hotspot of current tumor treatment and early disease diagnosis. At present, the preparation of the nano gold particles is divided into two strategies, namely a top-down physical method and a bottom-up chemical method. The seed growth method is the most common method at present, and can further grow the gold core into the gold nano-particles with specific size and shape.

In order to rapidly and efficiently obtain nanogold with different shapes and properties, which can be used in the fields of tumor diagnosis and treatment such as photothermal therapy, photoacoustic imaging and the like, a universal and environment-friendly preparation method of nanogold particles is developed to meet new research requirements.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to synthesize a series of gold nanoparticles with different shapes and sizes of 50nm or 100nm by using a seed growth method, and the obtained gold nanoparticles have better water solubility and monodispersity and good biocompatibility and can be used for tumor photothermal therapy and photoacoustic imaging.

In order to achieve the above purpose, the invention provides the following technical scheme:

in a first aspect, a method for preparing gold nanoparticles with controllable shape and size is provided, which comprises the following steps:

(1) preparation of seed solution: dissolving a proper amount of stabilizer in deionized water, adding chloroauric acid, stirring uniformly, then adding a sodium borohydride solution prepared by ice water or adopting a thermal reduction mode to form a brown yellow or red solution, and keeping the temperature of 25-110 ℃ for reaction;

(2) preparation of growth solution: a. dissolving a certain amount of stabilizer in deionized water, mixing with chloroauric acid solution, and changing the solution from dark yellow to colorless;

or,

a', dissolving a certain amount of stabilizer in deionized water, mixing with chloroauric acid solution, and then adding a structure directing agent, a pH regulator and a reducing agent to change the solution from dark yellow to colorless;

(3) mixing the seed solution obtained in the step (1) with the growth solution obtained in the step (2), and reacting in a water bath at 29-95 ℃;

or,

mixing the seed solution obtained in the step (1) with the growth solution obtained in the step (2) to prepare a new seed solution, mixing the new seed solution with the growth solution obtained in the step (2), and keeping the new seed solution in a water bath at 29-95 ℃ for reaction;

(4) and after the reaction is finished, collecting, centrifuging and purifying to obtain the nano gold particles.

Preferably, in the above preparation method, the stabilizer in step (1) and step (2) is cetyl trimethyl ammonium bromide or sodium citrate; the structure directing agent in the step (2) is silver nitrate or potassium iodide, the pH regulator is hydrochloric acid, and the reducing agent is ascorbic acid or hydroquinone.

Preferably, in the above preparation method, the final concentration of chloroauric acid is 0.01-0.025mol/L, the final concentration of sodium borohydride is 0.01-0.1mol/L, the final concentration of cetyltrimethylammonium bromide is 0.0001-0.1mol/L, the final concentration of silver nitrate is 0.004-0.01mol/L, the final concentration of potassium iodide is 0.01-0.1mol/L, the final concentration of hydroquinone is 0.01-0.06mol/L, the final concentration of ascorbic acid is 0.01-0.1mol/L, the final concentration of sodium citrate is 0.0022-0.02mol/L, and the final concentration of hydrochloric acid is 0.5-1.5 mol/L.

Preferably, in the above preparation method, the optimal seed reaction time in step (1) is 1-4h, and the optimal growth reaction time in step (3) is 12-16 h.

In a second aspect, the nano gold particles obtained by the preparation method are provided, the morphology of the nano gold particles is any one of spherical, cubic, star-shaped, rod-shaped, dumbbell and bipyramid, the particle size is 50nm or 100nm respectively, and the total number of the nano gold particles is 12.

The twelve types of nano gold particles prepared by the invention have obvious difference of maximum absorption peaks through UV/Vis wavelength scanning, the absorption peaks and colors show changes along with different shapes and sizes, and the maximum characteristic absorption peak of an ultraviolet-visible-infrared spectrum is positioned at 400-900 nm.

Zeta-potential results before and after centrifugal purification of the prepared gold nanoparticles show that after the centrifugal purification, after the excessive hexadecyl trimethyl ammonium bromide in the system is removed, potentials are reduced, but absolute values are still larger than 30mV, which shows that after the excessive hexadecyl trimethyl ammonium bromide in the system is removed by purification, the stability is good.

In a third aspect, the application of the gold nanoparticles in tumor photothermal therapy or tumor photoacoustic imaging is provided.

The present invention uses cetyl trimethyl ammonium bromide or sodium citrate as stabilizer, silver nitrate and potassium iodide as structure directing agent, ascorbic acid, sodium borohydride or hydroquinone as reducing agent, hydrochloric acid as pH regulator, and adopts two-step method to obtain a series of 50nm and 100nm star-shaped nano gold with spherical, rod-shaped, dumbbell-shaped, double cone-shaped, cubic and branched structures by changing the dosage of various compounds and the volume of the first-step seed solution.

Compared with the known method for synthesizing the nanogold material, the method has the advantages of simple and feasible preparation process, mild experimental conditions, cheap and easily-obtained required experimental reagents, effectively controlled morphology and size of the synthesized nanogold, good water solubility, good monodispersity, good stability, no toxic or side effect proved by in vitro experiments and good biocompatibility. Can be used for photothermal therapy, tumor imaging and the like.

Drawings

FIG. 1 shows the corresponding UV-Vis spectra of the nanogold solution with different morphologies of 50nm and 100nm in the embodiment.

FIG. 2 shows Zeta potentials before and after purification of 12 nanogold in a specific implementation method.

FIG. 3 shows transmission electron microscope images of 50nm and 100nm spherical nanogold described in example 1, wherein a is 50nm, b is 100 nm.

FIG. 4 shows transmission electron micrographs of 50nm and 100nm cubic nanogold as described in example 2, a, 50nm, b, and 100 nm.

FIG. 5 shows transmission electron micrographs of star-shaped 50nm and 100nm nanogold as described in example 3, a, 50nm, b, and 100 nm.

FIG. 6 is the transmission electron microscope pictures of the rod-shaped nanogold of 50nm and 100nm described in example 4, a, 50nm, b, and 100 nm.

FIG. 7 is a transmission electron micrograph of 50nm and 100nm dumbbell-shaped nanogold, a, 50nm, b, 100nm, described in example 5.

FIG. 8 is transmission electron microscope pictures of 50nm and 100nm bipyramidal nanogold described in example 6, a, 50nm, b, 100 nm.

Fig. 9 shows the actual particle size of nanogold through TEM in all examples.

FIG. 10 shows the toxic effect of six nanogold at 50nm on HepG2 and LO2 cells in all examples.

FIG. 11 shows the toxic effect of six nanogold at 100nm on HepG2 and LO2 cells in all examples.

Fig. 12 is a photo-thermal imaging diagram of 100nm bipyramid nanogold during tumor photo-thermal treatment.

Fig. 13 is a tumor ultrasonic-photoacoustic imaging picture of 100nm cubic nano-gold.

Detailed Description

The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.

The concentrations in the following examples are all final concentrations.

Example 1: synthesis of 50nm and 100nm spherical nano gold

1) Preparation of seeds

Weighing a certain amount of sodium citrate trihydrate, dissolving the sodium citrate trihydrate in 150mL of first-grade water to prepare 2.2mmol/L, and adding HAuCl₄(concentration 25mmol/L) the solution was stirred vigorously in a water bath at 110 ℃ for 15 min. The color of the solution was observed to change from yellow to blue-gray, then to pink within 10min, to wine-red in about 30 min.

2) Preparation of growth solution

53mL of primary water, 2mL of sodium citrate (60mmol/L) and HAuCl were taken₄Preparing solution from (25mmol/L), heating to 90 deg.C, cooling to 90 deg.C, and making into seed solution55mL of the solution was taken out and mixed with the reaction solution, and the mixture was reacted in a water bath at 95 ℃ for a certain period of time. The size of the nano gold particles is increased along with the increase of the size of the nano gold particles, and spherical nano gold with the particle size of 50nm and 100nm can be formed.

Example 2: synthesis of 50nm and 100nm cubic nanogold

1) Preparation of 50nm cubic seeds

First, HAuCl is added₄(0.01mol/L) solution and cetyltrimethylammonium bromide were added to 10mL of primary water and shaken well for 5min, and the color of the solution was observed to change from light yellow to orange. Then adding the prepared NaBH at 25 DEG C₄(0.1mol/L) solution. Upon addition, the color of the solution was observed to change from orange to brown immediately.

2) Preparation of 50nm cubic growth solution

HAuCl was added to the aqueous cetyl trimethyl ammonium bromide solution at room temperature₄(0.01 mol/L). Then, a freshly prepared aqueous solution of ascorbic acid (0.1mol/L) was added to the mixture solution, the color of the solution immediately changed from orange to colorless, and the prepared seeds were dropped into the colorless liquid using a 5. mu.L micro syringe, reacted for 12 to 14 hours, and the solution was seen to appear deep red.

3) Preparation of 100nm cubic seed ①

0.25mL of HAuCl₄(0.01mol/L) of the aqueous solution was added to a mixed solution of cetyltrimethylammonium bromide (0.1 mol/L). Shaking for several minutes, adding ice water to prepare NaBH₄(0.1mol/L) aqueous solution, and then stirred at 28 ℃ for 1h in the absence of light.

4) Preparation of 100nm cubic seed ②

The above gold seeds were diluted 10-fold and added to a solution containing 6mL of 0.1mol/L ascorbic acid, cetyltrimethylammonium bromide (0.1mmol/L) and HAuCl₄The growth was continued for 1h (0.01 mol/L).

5) Preparation of 100nm cubic growth solution

Seed ② was added to a solution containing cetyltrimethylammonium bromide, HAuCl₄(0.01mol/L) and ascorbic acid (0.1mol/L), the total volume of the solution was 10 mL. And reacting for 12-14h in the dark.

Example 3: synthesis of 50nm and 100nm star shaped nano gold

1) Preparation of seeds

Adding HAuCl₄(0.01mol/L) and trisodium citrate (0.02mol/L) were added to the primary water, and the total volume of the mixed solution was 20 mL. Then adding 60 mu L of ice water prepared sodium borohydride NaBH under stirring₄Solution (0.1 mol/L). Adding NaBH₄The solution turns pink immediately after the reaction, and gold spherical seeds are formed after the reaction for 2 hours in a dark place.

2) Preparation of growth solution

Cetyl trimethyl ammonium bromide is weighed and dissolved in 9.5mL of primary water, and HAuCl is added₄(0.01 mol/L). The solution changed from yellow to orange. First adding AgNO under the condition of violent stirring₃(0.01mol/L), and ascorbic acid (0.1mol/L) is then added. The orange solution turned into a clear colorless solution. Then, a certain amount of the seed solution is added into the colorless solution for reaction for a certain time.

Example 4: synthesis of 50nm and 100nm rod-like nanogold

1) Preparation of 50nm Bar-shaped seeds

Cetyl trimethyl ammonium bromide solution (0.1mol/L) and 0.01mol/L HAuCl₄And (4) mixing. Adding ice water prepared 0.01mol/L NaBH into the stirred solution₄The solution formed a brownish yellow solution and vigorous stirring of the seed solution was continued. The reaction was maintained at 25 ℃ for 2 h.

2) Preparation of 50nm rod-like growth solution

Temperature maintenanceHAuCl was added constantly to a solution of cetyltrimethylammonium bromide (0.1mol/L) at 29 deg.C₄(0.01mol/L), mixing thoroughly, adding AgNO₃(0.004mol/L) solution. Then, ascorbic acid (0.0788mol/L) solution was added as a mild reducing agent. The growth solution changed from dark yellow to colorless. mu.L of the seed solution was added to the growth solution. The color of the solution is gradually changed within 10-20 minutes, and the rod-shaped nano gold with the particle size of 50nm is obtained after the reaction for a certain time.

3) Preparation of 100nm Bar seed ①

4) Preparation of 100nm gold stick seed ②

The temperature was kept constant at 29 ℃ by adding HAuCl to a solution of cetyltrimethylammonium bromide (0.1mol/L)₄(0.01mol/L), mixing thoroughly, adding AgNO₃(0.004mol/L) solution, ascorbic acid (0.0788mol/L) solution as a mild reducing agent was then added, the solution turned colorless from dark yellow, 12. mu.L of seed ① solution was added to the colorless solution, after a certain time of reaction, gold nanorod solution (10mL) was centrifuged at 8000rpm × 30 minutes to remove excess silver salt, ascorbic acid and HCl in the growth solution, and redispersed in 2mL of cetyltrimethylammonium bromide solution (0.1mol/L) so that the concentration of gold nanorods was 2.5mmol/L, yielding a seed ② solution for further growth.

5) Preparation of 100nm rod-shaped growth solution

In AgNO₃Secondary growth of gold nanorods in the presence of a solution of cetyltrimethylammonium bromide (0.1mol/L) and HAuCl in a total volume of 10mL₄(0.01mol/L) mixing to fully complex gold salt, and then adding AgNO₃(0.004mol/L) and ascorbic acid (0.1 mol/L). finally, a solution of gold nanorod seeds ② is added.

Example 5: synthesis of 50nm and 100nm dumbbell-shaped nanogold

1) Preparation of dumbbell seed solution ①

Cetyl trimethyl ammonium bromide solution (0.1mol/L) and 0.01mol/L HAuCl₄And (4) mixing. Adding ice water prepared 0.01mol/L NaBH into the stirred solution₄The solution formed a brown-yellow solution and continued to stir vigorously. The reaction was maintained at 25 ℃ for 2 h.

2) Preparation of dumbbell seed solution ②

The temperature was kept constant at 29 ℃ and HAuCl was added to a solution of cetyltrimethylammonium bromide (0.1mol/L)₄(0.01mol/L), mixing thoroughly, adding AgNO₃(0.004mol/L) solution, ascorbic acid (0.0788mol/L) as a mild reducing agent was then added, the growth solution turned from dark yellow to colorless, 12. mu.L of a seed ① solution was added to the above solution, and the seed solution ② was obtained by reaction.

3) Preparation of dumbbell growth liquid

Cetyl trimethyl ammonium bromide (0.1mol/L) was mixed with HAuCl₄(0.01mol/L) are fully mixed to form complex gold salt, then certain amount of KI (0.01mol/L) and ascorbic acid (0.1mol/L) are added, finally, solutions of seeds ② with different volumes are added, and the gold nanorods are secondarily grown into dumbbell-shaped nanogold with the size of 50nm and 100nm in the presence of KI.

Example 6: synthesis of 50nm and 100nm biconical nano-gold

1) Preparation of seeds

Adding HAuCl₄(0.01mol/L) was mixed with an aqueous solution of cetyltrimethylammonium bromide (0.1 mol/L). Adding freshly prepared NaBH₄(0.1mol/L) an ice-water solution. The solution changed from orange to brown indicating seed particle formation. Washing the seed solution in the dark at 40 deg.C for one week. The synthesis of bipyramid nanogold cannot be achieved using freshly prepared seeds, which need to be aged for at least 7 days.

2) Preparation of growth solution

Adding HAuCl₄(0.01mol/L) was added to an aqueous solution of cetyltrimethylammonium bromide (0.1 mol/L). AgNO added with stirring₃(4mmol/L) followed by hydroquinone (60mmol/L) and the appropriate volume of seed solution. Protected from light and water bath at 40 ℃ for 12 h.

Example 7: cytotoxicity

The twelve kinds of gold nanoparticles described in this example were synthesized by adjusting the amount of various additives using a seed growth method, the particle sizes were respectively concentrated at 50nm and 100nm, and the gold nanoparticles described in this example had good stability and biocompatibility, and the toxicity of the gold nanoparticles described in this invention on human hepatoma cells (HepG2) and normal hepatocytes (L02) was determined using the MTT method.

Example 8: 100nm bipyramid nanogold tumor photothermal therapy

Injecting 200 mu L PBS solution containing 100nm bipyramid nanogold 200 microgram into tumor-bearing mice inoculated with 4T1 mouse breast cancer cells at 500mW/cm in right hind limb through tail vein²Under the continuous irradiation of laser with the power of 808nm, a thermal imaging camera shoots a whole body imaging picture of the mouse, referring to the attached figure 12 of the specification, and the temperature of the tumor part of the tumor-bearing mouse reaches 65 ℃ after the laser irradiation is carried out for 10 minutes. Tumor size was monitored over a long period of time and mice were found to have completely disappeared tumors after laser irradiation. The material of the invention has better application prospect in the aspect of tumor photothermal treatment.

Example 9: photoacoustic imaging of 100nm cubic nanogold for tumors

50 mu L of PBS solution containing 50 mu g of cubic nanogold with the length of 100nm is injected into tumor-bearing mice with breast cancer cells of 4T1 mice inoculated on the right hind limb through tumor injection, a Vevo LAZR small animal photoacoustic imaging system is adopted to shoot an imaging picture of tumor parts of the mice, see the attached figure 13 of the specification, and the tumor parts of the mice show obvious photoacoustic signals after the nanogold is injected. The material of the invention has better application prospect in the aspect of tumor photoacoustic imaging.

Claims

1. A method for preparing gold nanoparticles with controllable shape and size is characterized by comprising the following steps:

or,

2. The method for preparing gold nanoparticles with controllable shape and size according to claim 1, wherein the stabilizer in step (1) and step (2) is cetyl trimethyl ammonium bromide or sodium citrate; the structure directing agent in the step (2) is silver nitrate or potassium iodide, the pH regulator is hydrochloric acid, and the reducing agent is ascorbic acid or hydroquinone.

3. The method of claim 2, wherein the final concentration of chloroauric acid is 0.01 to 0.025mol/L, the final concentration of sodium borohydride is 0.01 to 0.1mol/L, the final concentration of cetyltrimethylammonium bromide is 0.0001 to 0.1mol/L, the final concentration of silver nitrate is 0.004 to 0.01mol/L, the final concentration of potassium iodide is 0.01 to 0.1mol/L, the final concentration of hydroquinone is 0.01 to 0.06mol/L, the final concentration of ascorbic acid is 0.01 to 0.1mol/L, the final concentration of sodium citrate is 0.0022 to 0.02mol/L, and the final concentration of hydrochloric acid is 0.5 to 1.5 mol/L.

4. The method for preparing gold nanoparticles with controllable shape and size according to claim 1, wherein the optimal seed reaction time in step (1) is 1-4h, and the optimal growth reaction time in step (3) is 12-16 h.

5. The gold nanoparticles obtained by the preparation method according to any one of claims 1 to 4, wherein the gold nanoparticles have any one of a spherical shape, a cubic shape, a star shape, a rod shape, a dumbbell shape and a biconical shape, and have a particle diameter of 50nm or 100 nm.

6. Use of the gold nanoparticles of claim 5 for the preparation of a medicament for tumor photothermal therapy or tumor photoacoustic imaging.