Preparation method and application of silver sulfide nanomaterials with photothermal / photoacoustic properties

Abstract

This invention relates to the technical fields of biological, chemical, and medical materials, specifically to a method for preparing and applying silver-sulfur nanomaterials with photothermal / photoacoustic properties. The silver-sulfur nanomaterials are Ag₂Se or Ag₂Te. The preparation method utilizes yeast cells as a biosynthesizer and uses any one of soluble selenite, soluble selenate, soluble tellurite, or soluble tellurite as an additive to synthesize the material. The preparation method of this invention employs mild reaction conditions, avoids complex operations and toxic solvents, and achieves a photothermal conversion efficiency of up to 76% for Ag₂Se nanocrystals under 808 nm laser irradiation; the photothermal conversion efficiency of Ag₂Te nanocrystals reaches up to 64% under 1064 nm laser irradiation. Furthermore, the Ag₂Se and Ag₂Te nanomaterials exhibit superior photoacoustic signal response compared to the commercially available photoacoustic contrast agent ICG.

Description

Technical Field

[0001] This invention relates to the technical fields of biological, chemical and medical materials, specifically to a method for preparing and applying silver-sulfur nanomaterials with photothermal / photoacoustic properties. Background Technology

[0002] Photoacoustic imaging (PAI) is a non-invasive medical imaging technique that provides high-contrast, high-sensitivity, high-depth-resolution, and high-spatial-resolution images. PAI utilizes pulsed lasers as the excitation source, acquiring ultrasound waves generated by the thermal expansion of biological tissues, and ultimately reconstructing the light absorption distribution of the biological tissue using algorithms. Some light-absorbing substances in living organisms, such as hemoglobin, melanin, and lipids, can provide photoacoustic signals. Using exogenous materials can enable photoacoustic imaging of specific tissues, such as tumors. Therefore, developing effective exogenous photoacoustic imaging contrast agents is crucial for in vivo tumor photoacoustic imaging.

[0003] Silver-sulfur quantum dots (MSDs) are widely used in fluorescence imaging and biosensing due to their optical stability and narrow band gap. The dangling bonds on the surface of quantum dots enhance the nonradiative relaxation of excitons, thereby converting all or part of the absorbed light energy into heat. Therefore, quantum dots are a promising material for photoacoustic imaging. However, there are currently few reports on the application of MSDs in photoacoustic imaging.

[0004] Oil-phase synthesis methods commonly used to synthesize silver sulfide quantum dots typically involve high temperatures, high pressures, and toxic solvents. Further in vivo applications require water-soluble modification, which hinders their application in clinical medicine. Live-cell synthesis of nanomaterials utilizes intracellular metabolism, enabling material synthesis under mild conditions. Furthermore, the products obtained through this method possess a biomolecular coating, resulting in excellent water solubility and biocompatibility, allowing for in vivo application without further modification. Biosynthetic methods not only provide high-performance imaging materials but also meet the demands of green chemistry. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for preparing silver-sulfur nanomaterials with photothermal / photoacoustic properties. The prepared silver-sulfur nanomaterials are synthesized and extracted from cells, and therefore have many biomolecules on their surface, exhibiting good biocompatibility. They can be passively aggregated at the tumor site through the high permeability and retention effect of solid tumors, thereby achieving high-contrast photoacoustic imaging.

[0006] The second objective of this invention is to provide an application of silver-sulfur nanomaterials with photothermal / photoacoustic properties.

[0007] One of the solutions adopted to achieve the objective of this invention is: a method for preparing silver sulfide nanomaterials with photothermal / photoacoustic properties, wherein the silver sulfide nanomaterials are Ag2Se or Ag2Te, and the preparation method is to use yeast cells as a biosynthesizer and any one of soluble selenite, soluble selenate, soluble tellurite, and soluble tellurite as additives to achieve the synthesis of the material.

[0008] Preferably, the soluble selenite is Li2SeO3, NaHSeO3, Na2SeO3, K2SeO3, KHSeO3, Cs2SeO3, or (NH4)2SeO3; the soluble selenate is Li2Se, Na2SeO4, K2SeO4, Cs2SeO4, or (NH4)2SeO4; the soluble tellurite is Li2TeO3, Na2TeO3, K2TeO3, or (NH4)2TeO3; and the soluble tellurite is Na2TeO4, K2TeO4, Cs2TeO4, or (NH4)2TeO4.

[0009] Preferably, the method includes the following steps:

[0010] (1) After inoculating and incubating the yeast strain to obtain colonies, continue incubation in the culture medium until the cells regain their viability to obtain activated bacterial solution;

[0011] (2) Inoculate the activated bacterial solution obtained in step (1) and culture it until the yeast cells are in the quiescent phase of growth;

[0012] (3) Using the cells obtained in step (2) as biosynthesizers, add any one of soluble selenite solution, soluble selenate solution, soluble tellurite solution, or soluble tellurite solution as additives, and co-incubate to obtain cells that are rich in selenium or tellurium.

[0013] (4) Using the obtained selenium-rich or tellurium-rich cells as biosynthesizers, add soluble silver salt solution to them and co-incubate to obtain yeast cells with nanocrystals inside the cells.

[0014] (5) Separate the bacterial precipitate from the bacterial suspension system in step (4), wash the bacterial precipitate and break it up; centrifuge the broken suspension to remove the precipitate and take the supernatant; digest the supernatant completely with proteolytic enzyme and concentrate it; purify the concentrate, remove the protein, cut off the gel strip with the product, and dialyze to recover the product, thus obtaining the silver-sulfur nanomaterial.

[0015] Preferably, in step (1), after the yeast strain is inoculated into YPD solid medium and incubated to obtain colonies, it is incubated in YPD liquid medium until the cells regain their viability. The pH value of YPD medium is 7.0-8.5 and the culture temperature is 20-30℃.

[0016] Preferably, in step (2), the activated bacterial solution is inoculated into fresh YPD liquid culture medium for cultivation.

[0017] Preferably, in step (3), the final concentration of the soluble selenite, soluble selenate, soluble tellurite, or soluble tellurite after addition is 1–10 mmol / L, and the co-incubation conditions are 20–30°C, 150–200 rpm in a shaker for 12–48 h.

[0018] Preferably, in step (4), the final concentration of the soluble silver salt after addition is 0.5-1 mmol / L, and the co-incubation conditions are 20-30°C, 150-200 rpm in a shaker for 12-48 h. The soluble silver salt is at least one of AgF, AgNO3, and AgClO4.

[0019] Preferably, in step (5), the final concentration of the proteolytic enzyme is 0.15–0.45 mg / mL, and the proteolytic enzyme is any one of trypsin, chymotrypsin, pepsin, thermophilic protease, staphylococcal protease, clostridium protease, and proteinase K.

[0020] Preferably, in step (5), purification is performed by agarose gel electrophoresis, using a sodium carbonate-sodium bicarbonate aqueous solution of 5-10 mmol / L and pH 9.0-10.0 as the electrophoresis buffer.

[0021] The second objective of this invention is achieved through the application of Ag2Se nanomaterials with photothermal / photoacoustic properties prepared by the aforementioned method in photoacoustic imaging.

[0022] The present invention has the following advantages and beneficial effects:

[0023] The preparation method of this invention has mild reaction conditions, does not involve complex operations or toxic solvents, and the synthesis process and products have little environmental pollution. The synthesized products can be further applied to living organisms without further modification, which has significant guiding significance for the synthesis of medical materials.

[0024] The silver-sulfur nanomaterials prepared by the method of this invention possess photothermal / photoacoustic properties, good biocompatibility, and are safe and non-toxic. The photothermal conversion efficiency of the prepared Ag₂Se nanocrystals can reach up to 76% under 808nm laser irradiation; the photothermal conversion efficiency of the Ag₂Te nanocrystals can reach up to 64% under 1064nm laser irradiation. Furthermore, the Ag₂Se and Ag₂Te nanomaterials exhibit better photoacoustic signal response than the commercially available photoacoustic contrast agent ICG.

[0025] The silver-sulfur nanomaterials prepared by the method of the present invention can be further applied to in vivo tumor imaging in mice to obtain high-contrast photoacoustic imaging images. Attached Figure Description

[0026] Figure 1 Transmission electron microscope images of (a) Ag2Se nanocrystals and (b) Ag2Te nanocrystals prepared in this invention.

[0027] Figure 2 XRD patterns of (a) Ag2Se nanocrystals and (b) Ag2Te nanocrystals prepared by this invention.

[0028] Figure 3 The heating and cooling curves of (a) Ag2Se nanocrystals and (b) Ag2Te nanocrystals prepared by this invention, as well as the linear fitting curve of time versus -lnθ.

[0029] Figure 4 In vitro photoacoustic imaging images of the Ag2Se nanocrystals prepared by this invention and the commercial contrast agent ICG.

[0030] Figure 5 Photoacoustic imaging of the Ag2Se nanocrystals prepared in this invention before and after tail vein injection into 4T1 tumor-bearing nude mice. Detailed Implementation

[0031] To better understand the present invention, the following embodiments are further illustrations of the present invention, but the content of the present invention is not limited to the following embodiments.

[0032] Example 1:

[0033] I. Preparation of Ag₂Se nanocrystals using yeast cells

[0034] 1) Yeast strains were inoculated onto YPD solid medium by streak plating and incubated in a constant temperature shaker (25℃) for 48 h to obtain yeast colonies;

[0035] 2) Transfer the single colony from step 1) to an Erlenmeyer flask (40 mL volume) containing 10 mL of YPD liquid medium, and incubate in a constant temperature shaker (25℃, 180 rpm) for 24 h until the cells regain viability;

[0036] 3) Inoculate the activated bacterial solution obtained in step 2) into an Erlenmeyer flask (250 mL volume) containing 100 mL of YPD liquid culture medium at an inoculation ratio of 2:100, and incubate for 24 h.

[0037] 4) Using the bacterial culture obtained in step 3) as a biosynthesizer, sodium selenate solution (final concentration 2 mmol / L) was added to it, and the culture was incubated in a constant temperature shaker (25℃, 180 rpm) for 24 h to obtain a yeast culture with intracellular "selenium-enriched" cells; the culture was then replaced with fresh YPD liquid culture medium, and the "selenium-enriched" yeast cells were used as a biosynthesizer. Silver nitrate solution (final concentration 0.5 mmol / L) was added to it, and the culture was incubated in a constant temperature shaker (25℃, 180 rpm) for 12 h to obtain a yeast culture with intracellular Ag2Se nanoparticles;

[0038] 5) Wash the bacterial suspension obtained in step 4) three times with ultrapure water, resuspend the bacterial precipitate in ultrapure water, and then use an ultrasonic cell disruptor to disrupt the bacterial suspension for 15 min. Centrifuge the disrupted suspension twice at 10000 rpm for 20 min, remove the precipitate and take the supernatant. After filtering through 0.22 μm, digest with trypsin for 2 h. After ultrafiltration and concentration of the digest, purify the concentrate by agarose gel electrophoresis to remove the protein in the concentrate. Then cut off the gel band with the product and dialyze the product back to the product using an 8000-14000 Da dialysis bag. The buffer used is 5 mmol / L pH 10.0 sodium carbonate-sodium bicarbonate buffer solution.

[0039] Figure 1 (a) is a transmission electron microscope image of the Ag2Se nanocrystals prepared in this embodiment. As can be seen from the figure, the prepared Ag2Se nanocrystals have good dispersibility.

[0040] Figure 2 (a) is the XRD pattern of the Ag2Se nanocrystals prepared in this embodiment, which matches the standard PDF card (PDF#24-1041) for orthorhombic Ag2Se nanocrystals.

[0041] Figure 3 (a) shows the heating and cooling curves of Ag2Se nanocrystals prepared by yeast in this invention. The photothermal conversion efficiency of Ag2Se nanocrystals at 808 nm is 76% calculated from the linear fitting curve of the cooling stage time and -lnθ.

[0042] Figure 4 The images show in vitro photoacoustic imaging of the prepared Ag2Se nanocrystals and the commercial contrast agent ICG. As can be seen from the images, the prepared Ag2Se nanocrystals have a better response to photoacoustic signals than the commercial contrast agent ICG, indicating that the prepared Ag2Se nanocrystals have excellent photoacoustic imaging performance.

[0043] II. Testing the in vivo photoacoustic imaging effect of the Ag2Se nanocrystals prepared in this example

[0044] First, Ag2Se nanocrystals were dispersed in 1×PBS. Then, the Ag2Se aqueous solution in PBS was injected into the nude mice via the tail vein of 4T1 tumor-bearing mice. Finally, the photoacoustic images of the tumor site before and after injection were observed using a small animal photoacoustic imaging device.

[0045] Figure 5 It is an observed image photograph, by Figure 5 It is evident that after injection of Ag2Se nanocrystals, a significant enhancement of photoacoustic signal was observed at the tumor site, indicating that Ag2Se nanocrystals can effectively perform in vivo tumor imaging.

[0046] Example 2:

[0047] I. Preparation of Ag2Te nanocrystals using yeast cells

[0048] 1) Yeast strains were inoculated onto YPD solid medium by streak plating and incubated in a constant temperature shaker (25℃) for 48 h to obtain yeast colonies;

[0049] 2) Transfer the single colony from step 1) to an Erlenmeyer flask (40 mL volume) containing 10 mL of LB liquid medium, and incubate in a constant temperature shaker (25℃, 180 rpm) for 24 h until the cells regain viability;

[0050] 3) Inoculate the activated bacterial solution obtained in step 2) into an Erlenmeyer flask (250 mL volume) containing 100 mL of YPD liquid culture medium at an inoculation ratio of 2:100, and incubate for 24 h.

[0051] 4) Using the bacterial culture obtained in step 3) as a biosynthesizer, sodium tellurate solution (final concentration 1 mmol / L) was added to it, and the culture was incubated in a constant temperature shaker (25℃, 180 rpm) for 24 h to obtain a yeast culture with intracellular "tellurium-rich" cells; the culture was then replaced with fresh YPD liquid medium, and the "tellurium-rich" yeast cells were used as a biosynthesizer. Silver nitrate solution (final concentration 0.5 mmol / L) was added to it, and the culture was incubated in a constant temperature shaker (25℃, 180 rpm) for 12 h to obtain a yeast culture with intracellular Ag2Te nanoparticles;

[0052] 5) Wash the bacterial suspension obtained in step 4) three times with ultrapure water, resuspend the bacterial precipitate in ultrapure water, and then use an ultrasonic cell disruptor to disrupt the bacterial suspension for 15 min. Centrifuge the disrupted suspension twice at 10000 rpm for 20 min, remove the precipitate and take the supernatant. After filtering through 0.22 μm, digest with trypsin for 2 h. After ultrafiltration and concentration of the digest, purify the concentrate by agarose gel electrophoresis to remove the protein in the concentrate. Then cut off the gel band with the product and dialyze the product back to the product using an 8000-14000 Da dialysis bag. The buffer used is 5 mmol / L pH 10.0 sodium carbonate-sodium bicarbonate buffer solution.

[0053] Figure 1 (b) is a transmission electron microscope image of the Ag2Te nanocrystals prepared in this embodiment. As can be seen from the figure, the prepared Ag2Te nanocrystals have good dispersibility.

[0054] Figure 2 (b) is the XRD pattern of the Ag2Te nanocrystals prepared in this embodiment, which matches the standard PDF card ((PDF#34-0142) for monoclinic Ag2Te nanocrystals).

[0055] Figure 3 (b) shows the heating and cooling curves of Ag2Te nanocrystals prepared by yeast in this invention. The photothermal conversion efficiency of Ag2Te nanocrystals at 1064 nm is 64%, which is calculated from the linear fitting curve of the cooling stage time and -lnθ.

[0056] The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing silver-sulfur nanomaterials with photothermal / photoacoustic properties, characterized in that: The silver-sulfur nanomaterial is Ag2Te, and its preparation method involves using yeast cells as a biosynthesizer and soluble tellurate as an additive to synthesize the material.

2. The method for preparing silver-sulfur nanomaterials with photothermal / photoacoustic properties according to claim 1, characterized in that: The soluble tellurate is Na2TeO4, K2TeO4, Cs2TeO4, or (NH4)2TeO4.

3. The method for preparing silver-sulfur nanomaterials with photothermal / photoacoustic properties according to claim 1 or 2, characterized in that, Includes the following steps: (1) After inoculating and incubating the yeast strain to obtain colonies, continue incubation in the culture medium until the cells regain their viability to obtain activated bacterial solution; (2) Inoculate and culture the activated bacterial solution obtained in step (1) until the yeast cells are in the quiescent phase of growth; (3) Using the cells obtained in step (2) as biosynthesizers, add any one of the soluble tellurate solutions as additives and co-incubate to obtain cells rich in intracellular tellurium. (4) Using the obtained tellurium-rich cells as biosynthesizers, add soluble silver salt solution to them and co-incubate to obtain yeast cells with nanocrystals inside the cells; (5) Separate the bacterial precipitate from the bacterial suspension system in step (4), wash the bacterial precipitate and break it up; centrifuge the broken suspension to remove the precipitate and take the supernatant; digest the supernatant completely with proteolytic enzyme and concentrate it; purify the concentrate, remove the protein, cut off the gel strip with the product, and dialyze to recover the product, thus obtaining the silver-sulfur nanomaterial.

4. The method for preparing silver-sulfur nanomaterials with photothermal / photoacoustic properties according to claim 3, characterized in that: In step (1), the yeast strain is inoculated into YPD solid medium and incubated until the colonies are obtained. Then, it is incubated in YPD liquid medium until the cells regain their viability. The pH value of YPD medium is 7.0~8.5 and the culture temperature is 20~30℃.

5. The method for preparing silver-sulfur nanomaterials with photothermal / photoacoustic properties according to claim 3, characterized in that: In step (2), the activated bacterial solution is inoculated into fresh YPD liquid culture medium for culture.

6. The method for preparing silver-sulfur nanomaterials with photothermal / photoacoustic properties according to claim 3, characterized in that: In step (3), the final concentration of the soluble tellurate after addition is 1~10 mmol / L, and the co-incubation conditions are 20~30℃, 150~200 rpm in a shaker for 12~48 h.

7. The method for preparing silver-sulfur nanomaterials with photothermal / photoacoustic properties according to claim 3, characterized in that: In step (4), the final concentration of the soluble silver salt after addition is 0.5~1 mmol / L, and the co-incubation conditions are 20~30℃, 150~200 rpm in a shaker for 12~48 h. The soluble silver salt is at least one of AgF, AgNO3, and AgClO4.

8. The method for preparing silver-sulfur nanomaterials with photothermal / photoacoustic properties according to claim 3, characterized in that: In step (5), the final concentration of the proteolytic enzyme is 0.15~0.45 mg / mL, and the proteolytic enzyme is any one of trypsin, chymotrypsin, pepsin, thermophilic protease, staphylococcal protease, clostridium protease, and proteinase K.

9. The method for preparing silver-sulfur nanomaterials with photothermal / photoacoustic properties according to claim 3, characterized in that: In step (5), purification is performed by agarose gel electrophoresis. The agarose gel electrophoresis solution is a sodium carbonate-sodium bicarbonate aqueous solution with a concentration of 5-10 mmol / L and a pH of 9.0-10.

0.

10. A method for preparing Ag₂Se nanocrystals using yeast cells, characterized in that: 1) Yeast strains were inoculated onto YPD solid medium by streak plating and incubated in a shaker at 25°C for 48 h to obtain yeast colonies; 2) Transfer the single colony from step 1) to an Erlenmeyer flask containing 10 mL of YPD liquid medium, place it in a constant temperature shaker, and incubate at 25°C and 180 rpm for 24 h until the cells regain viability; 3) Inoculate the activated bacterial solution obtained in step 2) into an Erlenmeyer flask containing 100 mL of YPD liquid culture medium at an inoculation ratio of 2:100 and incubate for 24 h; 4) Using the bacterial culture obtained in step 3) as a biosynthesizer, sodium selenate solution was added to it to a final concentration of 2 mmol / L, and the culture was incubated in a constant temperature shaker at 25℃ and 180 rpm for 24 h to obtain a yeast culture with intracellular selenium-enriched cells; fresh YPD liquid culture medium was then used, and the selenium-enriched yeast cells were used as a biosynthesizer. Silver nitrate solution was added to it to a final concentration of 0.5 mmol / L, and the culture was incubated in a constant temperature shaker at 25℃ and 180 rpm for 12 h to obtain a yeast culture with intracellular Ag2Se nanoparticles; 5) Wash the bacterial suspension obtained in step 4) three times with ultrapure water, resuspend the bacterial precipitate in ultrapure water, and then use an ultrasonic cell disruptor to disrupt the bacterial suspension for 15 min. Centrifuge the disrupted suspension twice at 10,000 rpm for 20 min, remove the precipitate and collect the supernatant. After filtering through 0.22 μm, digest with trypsin for 2 h. After ultrafiltration and concentration of the digest, purify the concentrate by agarose gel electrophoresis to remove the protein in the concentrate. Then cut off the gel band with the product and dialyze the product back to the product using an 8000-14000 Da dialysis bag. The buffer used is 5 mmol / L pH 10.0 sodium carbonate-sodium bicarbonate buffer solution.

Images