CN112775432B

CN112775432B - Short-wave infrared fluorescent gold nanocluster based on bovine serum albumin and preparation method and application thereof

Info

Publication number: CN112775432B
Application number: CN201911011485.3A
Authority: CN
Inventors: 肖玉玲; 洪学传; 唐琳; 曾小东
Original assignee: SUZHOU Institute OF WUHAN UNIVERSITY
Current assignee: SUZHOU Institute OF WUHAN UNIVERSITY
Priority date: 2019-10-23
Filing date: 2019-10-23
Publication date: 2023-04-18
Anticipated expiration: 2039-10-23
Also published as: CN112775432A

Abstract

The invention discloses a short-wave infrared fluorescence gold nanocluster based on bovine serum albumin and a preparation method and application thereof. Takes chloroauric acid as raw material, and adopts lysine, histidine and Au on bovine serum albumin ³⁺ Coordination of (2) and use of solution pH>Tyrosine and sodium borohydride on bovine serum albumin at 10 ℃ on Au ³⁺ The method is simple, reaction conditions are mild, the particle size of the prepared BSA @ AuNCs is less than 2nm, and the prepared BSA @ AuNCs has good water solubility and stability, good light stability, high fluorescence quantum yield and good biocompatibility. In addition, the BSA @ AuNCs synthesized by the invention can generate active oxygen under the laser radiation of 808nm, and can be used for tumor photodynamic therapy; the maximum fluorescence emission peak is about 915nm, can be used for short-wave infrared fluorescence imaging of tumors, and has excellent application prospect.

Description

Short-wave infrared fluorescent gold nanocluster based on bovine serum albumin and preparation method and application thereof

Technical Field

The invention belongs to the technical field of nano material preparation, relates to tumor imaging and photodynamic therapy in the field of biomedicine, and particularly relates to a bovine serum albumin gold nano cluster with short-wave infrared fluorescence emission, and a preparation method and application thereof.

Background

The tumor is a new organism formed by the continuous excessive proliferation of cells and the transformation of the cells due to the mutation and the abnormal function regulation of the gene level of the cells under the long-term synergistic action of internal and external tumorigenic factors, and has the characteristics of benign and malignant tumors, difficult treatment of the malignant tumor and extremely high death rate. In 2018, there were 1810 ten thousand new cancer cases and 960 ten thousand cancer death cases globally, among which, in china, there were 429.2 ten thousand new tumor cases and 281.4 ten thousand death cases. The lung cancer is the tumor with the highest morbidity and mortality in China, and the gastric cancer, the esophageal cancer and the liver cancer are the following common tumors with higher morbidity and mortality in China. The best strategy for treating tumors is early diagnosis, and the cure rate can be improved to 83 percent. The early diagnosis of the tumor is very important, the early treatment can be found early, the death rate is greatly reduced, and the statistical data shows that the cure rate can be improved to 83 percent by realizing the early diagnosis of the tumor. The development of new tumor-targeted imaging agents and drug delivery systems has become one of the important strategies for early diagnosis and treatment of tumors.

The application of the nano material in the field of life science provides a more effective method for early diagnosis and treatment of tumors. In the field of nano imaging materials, fluorescent Quantum Dots (QDs) are currently studied enthusiastically, and have many excellent optical, electronic and physicochemical properties, such as large Stokes shift, good light stability, wide excitation spectrum, adjustable emission wavelength, and narrow and symmetrical spectrum, which has attracted extensive attention of researchers in the field of early diagnosis of tumors. However, QDs also have some inherent disadvantages, such as containing toxic heavy metal elements, etc., which limit their potential clinical applications. Fluorescent noble metal Nanoclusters (NCs) are representative of new fluorescent nanomaterials in recent years, mainly refer to luminescent nanomaterials with a size of less than 2nm, which are composed of several to several tens of metal atoms of gold, silver, platinum and the like, and have unique optical, electronic and chemical properties due to the fact that the size of the luminescent nanomaterials is close to the Fermi (Fermi) wavelength of electrons. Compared with quantum dots, NCs have the advantages of low toxicity, ultrafine size and the like, and have high attention in tumor imaging and treatment.

Gold nanoclusters (AuNCs), unlike gold nanomaterials such as gold nanorods and the like, do not have a surface plasmon resonance effect, but have fluorescence in the visible-near infrared region, a characteristic mainly attributed to the fact that electron transition of sub-nanometer size of gold nanoclusters is smaller than Fermi wavelength. As a novel fluorescent nano material, compared with fluorescent materials such as fluorescent protein, fluorescent quantum dots and organic dyes, auNCs has excellent properties, such as stable chemical properties, good light stability, good photobleaching resistance, long fluorescence life, small size, large Stokes shift, adjustable emission spectrum, mild preparation conditions, simple preparation process, easy metabolism, good biocompatibility and the like, and has wide application prospect in the biomedical fields of cell and living body imaging, tumor diagnosis and treatment integration and the like. The protein is a biological macromolecule formed by condensing various amino acids, and AuNCs can be synthesized by using the protein as a modifier and a stabilizer, because the protein structure contains abundant functional groups such as carboxyl, amino, sulfydryl, phenolic hydroxyl and the like, and the functional groups and gold atoms have stronger interaction force, so that the AuNCs can be stabilized. Bovine Serum Albumin (BSA) is a globulin in bovine serum, contains 607 amino acid residues, has a molecular weight of 66.446KDa, has wide application in biochemical experiments and biomedicine, has been reported to be used as a modifier and a stabilizer to synthesize AuNCs, and the fluorescence spectrum of the conventional AuNCs is between 400 and 900 nm. In recent years, short-wave infrared (900 nm-1700 nm) imaging is more and more focused due to high penetration depth, high sensitivity and high resolution, and no report of short-wave infrared fluorescence gold nanocluster using bovine serum albumin as a modifier exists at present.

Therefore, the invention takes bovine serum albumin as a modifier, is used as a reducing agent and a protective agent in the synthesis process of AuNCs, and utilizes tyrosine and sodium borohydride to react Au ³⁺ Reducing property, obtaining short-wave infrared fluorescent gold nanocluster with fluorescence emission spectrum of 900nm-1700nm, and simultaneously, the gold nanocluster can generate active oxygen under 808nm laser radiation, so that biological tissue short-wave infrared imaging and tumor photodynamic therapy can be realized.

Disclosure of Invention

The invention aims to synthesize an ultra-small gold nanocluster by using the biocompatibility of bovine serum albumin, which has short-wave infrared fluorescence and generates active oxygen under the radiation of infrared light, can expand the fluorescence spectrum range of the bovine serum albumin gold nanocluster in the current research, can be applied to deep biological tissue imaging and background signal reduction, is also applied to tumor photodynamic therapy, and fills the blank of the existing bovine serum albumin gold nanocluster on the aspect.

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

(1) Accurately weighing bovine serum albumin, placing into a round-bottom flask treated with aqua regia in advance, adding primary water to prepare an aqueous solution, adding magnetons, and placing into a water bath kettle for stirring;

(2) Adding a chloroauric acid solution into the flask obtained in the step (1) under the stirring state, and adding a sodium hydroxide solution to adjust the pH value, so that the solution is in a clear state;

(3) Slowly adding a sodium borohydride solution prepared by ice water into the round-bottom flask in the step (2) and continuously stirring for reaction;

(4) And after the reaction is finished, purifying and concentrating the product by adopting an ultrafiltration centrifugal tube through an ultrafiltration centrifugal method to obtain the short-wave infrared fluorescence gold nanocluster.

In the step (1), the final concentration of the bovine serum albumin aqueous solution is 4-20mg/mL, and the temperature of the water bath is 25 ℃.

In the above step (2), HAuCl ₄ The final concentration of the solution is 0.01-0.05mol/L, the final concentration of the NaOH solution is 0.1-1mol/L, and the pH value of the reaction system is 10-12.

In the step (3), naBH ₄ The final concentration of the aqueous solution is 0.1-1mol/L, the reaction stirring temperature is 25-50 ℃, and the reaction time is 12-24h.

In the step (4), the cut-off molecular weight of the ultrafiltration centrifugal tube is 3-100kDa.

In the preparation method, the fluorescence intensity of the gold nanoclusters can be controlled by adjusting the dosage of various additives, the reaction temperature and the reaction time.

The invention provides application of the gold nanocluster in the fields of tumor imaging and treatment. The fluorescent probe has stronger emission spectrum in a short wave infrared region, and can be used for short wave infrared fluorescence imaging; in addition, it can generate active oxygen under near infrared radiation, and can be used for photodynamic therapy.

Compared with the prior art, the invention has the following beneficial effects:

(1) The preparation method of the gold nanocluster is simple and easy to operate, mild in condition, strong in controllability and good in yield.

(2) According to the gold nanocluster, due to the introduction of bovine serum albumin with good biocompatibility as a modifier and the adoption of a milder method for synthesis, the product has the characteristics of small nanometer size (less than 2 nm), good water solubility, uniform particle size, negligible biological toxicity and the like.

(3) The synthesis of the gold nanocluster of the invention introduces a reducing agent, namely sodium borohydride, on the traditional preparation method of the bovine serum albumin gold nanocluster, so that the fluorescence emission wavelength of the gold nanocluster is red-shifted from a visible light and near infrared region to a short wave infrared region, and the signal-to-noise ratio, the penetration depth and the resolution of biological tissue imaging are favorably improved.

(4) Compared with the traditional gold nanocluster, the gold nanocluster provided by the invention has a photodynamic effect and can be used for photodynamic treatment of tumors.

Drawings

FIG. 1 is a photograph of the gold nanoclusters of example 1 under fluorescent light (left) and short wave infrared fluorescence imager (right, 808nm laser excitation).

Fig. 2 is a transmission electron microscope image of the gold nanoclusters described in example 1.

Fig. 3 is an actual particle size (left) of the gold nanoclusters described in example 1 as exhibited by a transmission electron microscope and a hydrated particle size distribution (right) as shown by a nano-particle sizer.

Fig. 4 is an ultraviolet absorption spectrum and a fluorescence emission spectrum of the gold nanoclusters described in example 1.

FIG. 5 is the results of cytotoxicity tests of gold nanoclusters described in example 1 on L02, U87-MG and 4T1 cells.

Fig. 6 shows the results of the in vitro photodynamic test of gold nanoclusters as described in example 1.

Fig. 7 is the result of photodynamic therapy of 4T1 tumor cells by gold nanoclusters as described in example 1.

FIG. 8 is the short wave infrared imaging effect of the gold nanoclusters described in example 1 on tumors in 4T1 tumor-bearing mice.

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. Example 1 Synthesis of gold nanoclusters

The fluorescence intensity of the gold nanoclusters was controlled by adjusting the amount of each additive, the reaction temperature and the reaction time, and the specific orthogonal design factor levels are shown in table 1.

TABLE 1

(1) Accurately weighing appropriate amount of bovine serum albumin, respectively placing into round-bottom flask (treated with aqua regia in advance), adding primary water to prepare 2.5mL aqueous solution (4,6 and 8 mg/mL), placing into magneton with appropriate size, placing round-bottom flask into water bath, and stirring;

(2) Adding a chloroauric acid aqueous solution (0.03 mol/L,0.0625 mL) into the round-bottom flask, adding a sodium hydroxide solution of 0.5mol/L (0.0325,0.05 and 0.065 mL) to adjust the pH to 10-11, and enabling the solution to be in a clear state;

(3) Slowly adding a 0.5mol/L sodium borohydride solution (0.025,0.0375 and 0.05 mL) prepared in ice water, and continuing to stir in a water bath at 30 ℃ for reaction (12, 18 and 24 hours);

(4) After the reaction is finished, purifying and concentrating the product by a 30kDa ultrafiltration centrifugal tube to obtain the brown gold nanoclusters which have short-wave infrared fluorescence emission and generate active oxygen under near infrared light radiation. The results and analysis of the orthogonal tests are shown in Table 2. Finally, selecting the factor with the highest fluorescence intensity, namely the serial number 7 as the optimal condition according to the orthogonal design, wherein the electron microscope display of the optimal condition is shown as the attached figure 1 in the specification. In the following examples, the gold nanoclusters prepared by controlling the amount of the additive, the reaction temperature, and the reaction time shown in the number 7 in table 1 were used as the study objects.

TABLE 2

[ example 2 ]

The BSA @ AuNCs prepared in example 1 is subjected to morphology characterization by using a transmission electron microscope, and the specific morphology of the nanoparticles is determined to be round particles, as shown in the attached figure 2 of the specification.

[ example 3 ] A method for producing a polycarbonate

The real particle size of the gold nanoclusters in the transmission electron microscope picture obtained in example 2 is calculated by using ImageJ software, and the hydrated particle size of the BSA @ AuNCs prepared in example 1 is measured by using a nanometer particle sizer, wherein the particle size distribution is shown in figure 3 of the specification, and the main particle size distribution is about 1.5 nm.

[ example 4 ]

The absorption spectrum and the fluorescence emission spectrum of the gold nanocluster synthesized in example 1 were measured by an ultraviolet-visible-near infrared spectrophotometer and a fluorescence spectrometer, and the obtained results are shown in fig. 4 of the specification, where the absorption characteristic peak of bsa @ auncs is 260nm, the maximum fluorescence emission peak is 915nm, and is located in the short-wave infrared region.

[ example 5 ] A method for producing a polycarbonate

To determine the cytotoxicity of the gold nanoclusters synthesized in example 1, the viability of brain glioma cells (U87-MG cells), breast cancer cells (4T 1 cells) and normal liver cells (L02 cells) incubated with bsa @ auncs was examined by the MTT method. In the cytotoxicity determination, three cells in logarithmic growth phase are inoculated into a 96-well plate, 100 mu L of the three cells are added into each well, and the inoculation density is 1 multiplied by 10 ⁴ A hole. Different concentrationsThe BSA @ AuNCs solution of (0,0.25,0.5,1,2.5,5, 10, 25, 50, 100. Mu.g/mL) was added to each well of different groups. After culturing for 24h in a carbon dioxide incubator, washing with PBS for three times, adding MTT into each well, continuing culturing for 4h, removing supernatant, adding DMSO solution, shaking for 10min, and detecting absorbance at 492nm by using a microplate reader. The test results are shown in the accompanying figure 5 of the specification, and the results show that the cell viability of the gold nanoclusters on U87-MG and 4T1 cells is not obviously inhibited with the increase of the concentration, but the cell viability is reduced in L02 cells but still higher than 80%, which indicates that the BSA @ AuNCs nanoparticles do not generate obvious cytotoxicity on two cancer cells and normal liver cells, namely the synthesized nanoparticles have good biocompatibility.

[ example 6 ]

To examine the singlet oxygen generating ability of the gold nanoclusters prepared in example 1, 3mL of DPBF (0.75M ethanol solution) was added to the cuvette, and 200. Mu.L of BSA @ AuNCs solution was mixed, and then the cuvette was placed under a dark condition using 1 W.cm ^-2 808nm laser radiation for 0,1,2,3,4,5min. A control solution of DPBF was prepared by replacing the BSA @ AuNCs solution with 200. Mu.L of primary water and treated under the same laser conditions. And scanning the change condition of the wavelength by using an ultraviolet-visible-near infrared spectrophotometer, recording the absorbance values of all groups at 410.5nm, and plotting the absorbance ratio of the DPBF at 410.5nm and the laser radiation time at 808nm according to the absorbance ratio of different radiation times without radiation, wherein the graph is shown in figure 6 in the specification. The result shows that the gold nanocluster can generate active oxygen under 808nm laser radiation.

[ example 7 ]

Cellular photodynamic therapy of bsa @ auncs prepared in example 1: taking 4T1 cells growing in logarithmic phase, adding the cells in 96-well plate at 1X 10 per ml ⁴ 100mL of the cell suspension was placed in a standard carbon dioxide cell incubator and incubated for 24h. The original medium was then discarded, replaced with medium containing different concentrations of gold nanoclusters as described in example 1, and placed into a cell culture incubator for further incubation for 24h. Discarding the culture medium containing the medicine after 24 hours, replacing the culture medium with a complete culture medium without the medicine, and irradiating with laserThe power density of the used shooting cells is 0,1 and 2W-cm ^-2 Irradiating the mixed solution for 5min by using laser at 808nm, not performing radiation treatment on a blank control group, and putting the mixed solution into a cell culture box to continue incubation for 24h after the radiation is finished. And (3) discarding the culture medium after the incubation is finished, replacing the culture medium with a culture medium containing MTT, discarding the liquid after the incubation is carried out for 4h, dissolving the formed blue solid by DMSO, placing the 96-well plate on an ELISA reader, reading the ELISA reader at the 492nm wavelength, and determining the absorbance value. The cell survival rate shown in figure 7 of the specification is calculated according to a corresponding formula of the cell survival rate. The result shows that the gold nanocluster material has a good photodynamic treatment effect on breast cancer cells in the horizontal plane of the cells.

[ example 8 ]

4T1 tumor cells growing in logarithmic phase are obtained by adopting a standard cell culture technology, and are injected into the right hind limb of a Balb/c mouse by an insulin syringe to establish a 4T1 breast cancer mouse model. 200 mug of gold nanocluster PBS solution (1000 mug/mL, 200 mug) is injected into a 4T1 mouse breast cancer tumor-bearing mouse through tail vein, a mouse tumor part imaging graph is shot by a short wave infrared fluorescence imager (Suzhou Yirui optical technology limited), the specific biological imaging effect is shown in the specification and figure 8, the mouse tumor part shows an obvious fluorescence signal after the injection of the gold nanocluster, and the difference between the tumor tissue and the surrounding normal tissue is obvious.

Claims

1. The short-wave infrared fluorescence gold nanocluster based on bovine serum albumin is characterized in that the gold nanocluster is prepared by the following specific steps:

(1) Accurately weighing 20mg bovine serum albumin, placing the bovine serum albumin in a round bottom flask which is treated by aqua regia in advance, adding primary water to prepare an aqueous solution with the concentration of 2.5mL being 8mg/mL, placing magnetons with a proper size into the aqueous solution, and placing the round bottom flask in a water bath kettle for stirring;

(2) Adding 0.0625mL aqueous solution with the concentration of 0.03mol/L chloroauric acid into the round-bottom flask obtained in the step (1), adding 0.0325 mL aqueous solution with the concentration of 0.5mol/L sodium hydroxide solution, adjusting the pH value to 10-11, and enabling the solution to be in a clear state;

(3) Slowly adding 0.05mL solution prepared by ice water and with the concentration of 0.5mol/L sodium borohydride solution, and continuously stirring in a water bath at 30 ℃ for reacting for 18 hours;

(4) After the reaction was complete, the concentrated product was purified by a 30kDa ultrafiltration centrifuge tube and was brown in color.

2. Use of gold nanoclusters according to claim 1 for the preparation of products for short wave infrared fluorescence imaging of tumors.

3. Use of gold nanoclusters according to claim 1 for the preparation of products for photodynamic therapy of tumors, wherein said short wave infrared fluorescent gold nanoclusters are capable of generating reactive oxygen species under laser radiation of wavelength 400 nm-1300 nm.