CN109986090B - Double-ligand gold nanoparticle aqueous solution and preparation method and application thereof - Google Patents
Double-ligand gold nanoparticle aqueous solution and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a double-ligand gold nanoparticle aqueous solution and a preparation method and application thereof. The preparation method specifically comprises the following steps: adding a hydrosulphonyl micromolecule compound A aqueous solution into a solvent, adding a chloroauric acid aqueous solution under the conditions of oil bath and stirring, stirring for reaction, cooling and dialyzing to obtain a single ligand gold nanoparticle aqueous solution; and adding the single-ligand gold nanoparticle aqueous solution into a mercapto small molecular compound B aqueous solution, stirring for reaction, and dialyzing to obtain the double-ligand gold nanoparticle aqueous solution. The synthetic method has the advantages of simple process, low energy consumption, low cost and easy large-scale production. The rate of generating singlet oxygen of the double-ligand gold nanoparticle aqueous solution synthesized by the method is at least increased by over 87% compared with that of generating singlet oxygen of a single ligand, and the double-ligand gold nanoparticle aqueous solution is shown to inhibit the growth of cancer cells more obviously in an in-vitro cancer cell model compared with the single-ligand gold nanoparticle aqueous solution.
Description
Technical Field
The invention belongs to the field of functional optical nano materials, and particularly relates to a dual-ligand gold nanoparticle aqueous solution and a preparation method and application thereof.
Background
With the increasing incidence and mortality of cancer worldwide, the development of effective cancer diagnosis and treatment reagents is urgent. In the chemotherapy process, chemotherapy drugs generally have serious side effects and high drug resistance on patients, the curative effect of the chemotherapy drugs does not reach the ideal effect, and the tumor recurrence and metastasis are common. In order to improve the therapeutic effect of patients, various therapeutic methods have been widely proposed, among which photodynamic therapy is a rapidly developing cancer treatment. A high-activity singlet oxygen substance is generated by a photosensitizer under the condition of the coexistence of oxygen and light, the structural damage of proteins, DNA and the like in cancer cells is promoted, and the apoptosis of the cancer cells is further triggered, and meanwhile, the photosensitizer does not have any influence on the cancer cells and normal cells under the condition of dark light. The photodynamic therapy has the characteristics of no drug resistance, small side effect, small damage to marginal tissues and the like, so that the method is a potential tumor treatment means and is gradually popularized.
At present, singlet oxygen for photodynamic therapy is mainly generated by a traditional organic small molecular photosensitizer, but the traditional organic small molecular photosensitizer has the defects of complex synthesis process, poor water solubility, low selectivity, high skin toxicity, easiness in enzyme degradation and the like, so that the wider clinical application of the traditional organic small molecular photosensitizer is greatly limited. In recent years, nanoscale optical materials such as metal nanoparticles, semiconductor quantum dots and the like are reported to have the ability of generating singlet oxygen, wherein ultra-small luminescent gold nanoparticles (AuNPs) have the advantages of simple synthesis, good water solubility, high biocompatibility, high tumor targeting efficiency, good stability and the like, and show great clinical application prospects in the aspect of clinical photodynamic therapy. The main problem of ultra-small luminescence AuNPs is that the efficiency of singlet oxygen generation is low, and the requirements of photodynamic therapy in clinical treatment are difficult to meet. Therefore, the development and design of AuNPs with the property of efficiently generating singlet oxygen have important scientific research and clinical application values.
Disclosure of Invention
In order to improve the efficiency of generating singlet oxygen by AuNPs, the invention provides a method which is simple and easy to operate and has obvious improvement effect. The invention mainly aims to provide a double-ligand gold nanoparticle aqueous solution.
The invention also aims to provide a preparation method of the double-ligand gold nanoparticle aqueous solution.
The invention further aims to provide application of the double-ligand gold nanoparticle aqueous solution.
The AuNPs with the double ligand surface functionalized prepared by the method can efficiently generate high-activity singlet oxygen under the illumination condition, and the singlet oxygen acts on cancer cells to cause the cancer cells to die. Therefore, the double-ligand AuNPs prepared by the invention has a wide application prospect in the field of clinical tumor treatment.
The method is realized by the following technical scheme:
a preparation method of a double-ligand gold nanoparticle aqueous solution comprises the following synthetic reaction formula:
the method specifically comprises the following steps:
(1) adding the sulfydryl micromolecule compound A into water to prepare a sulfydryl micromolecule compound A water solution;
(2) adding a hydrosulphonyl micromolecule compound A aqueous solution into a solvent, adding a chloroauric acid aqueous solution under the conditions of oil bath and stirring, continuously stirring for reaction, stopping the reaction when the fluorescence intensity of a reaction system reaches the strongest value and is not increased any more, cooling to room temperature, putting into a dialysis bag, dialyzing in water, and obtaining a single-ligand gold nanoparticle aqueous solution after the dialysis is finished;
(3) and (3) adding the aqueous solution of the single-ligand gold nanoparticles prepared in the step (2) into the aqueous solution of the mercapto small molecular compound B, stirring at room temperature for reaction, putting the mixture into a dialysis bag after the reaction is finished, dialyzing in water, and obtaining the aqueous solution of the double-ligand gold nanoparticles after the dialysis is finished.
Preferably, the molar concentration of the aqueous solution of the mercapto small molecule compound a in step (1) is 0.01mol/L to 0.20 mol/L.
Preferably, the mercapto small molecule compound a in step (1) has the following structure: R-SH, wherein R is alkyl or heterocycle containing nitrogen or oxygen atom.
Preferably, the thiol small molecule compound a in step (1) is one of polyethylene glycol monomethyl ether thiol, thiol polyethylene glycol amino, thiol polyethylene glycol carboxyl, glutathione, bovine serum albumin and mercaptopropionic acid.
Preferably, the molar ratio of the mercapto small molecule compound a to the chloroauric acid in the step (2) is 1: 1-8: 1, the final concentration of the chloroauric acid in the reaction system is 1-5 mmol/L.
Preferably, the solvent in step (2) is one of ethanol, methanol, tetrahydrofuran, acetonitrile and water, and more preferably water.
Preferably, the temperature of the oil bath in the step (2) is 25-95 ℃.
Preferably, the speed of the continuous stirring in the step (2) is 1000 rpm/min-1500 rpm/min.
Preferably, the room temperature in the step (2) is 4-37 ℃.
Preferably, the molecular weight cut-off of the dialysis bag in the step (2) is 3-10 kDa.
Preferably, the dialysis in step (2) is specifically: dialyzing for at least three times, wherein each time is not less than 6 h.
Preferably, the mercapto small molecule compound B in step (3) is one of mercaptoethylamine, mercaptobenzimidazole and mercaptoimidazole.
Preferably, the concentration of the aqueous solution of the mercapto small molecular compound B in the step (3) is 0.01mmol/L to 1 mmol/L.
Preferably, the molar ratio of the mercapto small molecular compound B in the step (3) to the gold atoms in the aqueous solution of the single-ligand gold nanoparticles is 0.1: 1-10: 1, more preferably in a molar ratio of 3: 1, the final concentration of gold atoms in the single ligand gold nano particle aqueous solution in the reaction system is 0.08 mmol/L-20 mmol/L.
Preferably, the stirring time in the step (3) is 10min to 48 hours, and more preferably 24 hours.
Preferably, the room temperature in the step (3) is 4-37 ℃.
Preferably, the molecular weight cut-off of the dialysis bag in the step (3) is 3-10 kDa.
Preferably, the dialysis in step (3) is specifically: dialyzing for at least three times, wherein each time is not less than 6 h.
The double-ligand gold nanoparticle aqueous solution is prepared by the preparation method of the double-ligand gold nanoparticle aqueous solution.
The application of the double-ligand gold nanoparticle aqueous solution in preparing a photosensitizer.
Compared with the prior art, the invention has the following advantages and technical effects:
(1) the preparation method is simple, easy to operate, low in time consumption and low in cost;
(2) the preparation method has wide selectivity, and can be implemented aiming at single ligand gold nanoparticle aqueous solutions such as amination, carboxylation and the like;
(3) compared with the single-ligand gold nanoparticle aqueous solution, the rate of generating singlet oxygen by the double-ligand gold nanoparticle aqueous solution prepared by the method is at least increased by 87%, and in-vitro experiments prove that the double-ligand gold nanoparticle aqueous solution generates singlet oxygen under the condition of visible light to kill cancer cells, so that the double-ligand gold nanoparticle aqueous solution has great potential of being applied to clinical photodynamic therapy.
Drawings
FIG. 1 is an excitation, emission and absorption spectra of the aqueous solution of monodiganded gold nanoparticles prepared in example 1.
FIG. 2 is a transmission electron microscope photograph of the aqueous solution of monodiganded gold nanoparticles prepared in example 1.
FIG. 3 is an X-ray photoelectron spectroscopy (XPS) chart of the aqueous solution of monodiganded gold nanoparticles prepared in example 1.
FIG. 4 is a comparison chart of fluorescence spectra of the aqueous solution of mono-ligand gold nanoparticles and the aqueous solution of bi-ligand gold nanoparticles prepared in example 1.
FIG. 5 is a TEM image of the aqueous solution of dual ligand gold nanoparticles prepared in example 1.
Fig. 6 is a Zeta potential diagram of the aqueous solution of mono-ligand gold nanoparticles and di-ligand gold nanoparticles synthesized in example 1 under the condition of pH 7.4.
FIG. 7 is a UV-VIS absorption spectrum of 0.1mmol/L aqueous solution of monodiganded gold nanoparticles prepared in example 1.
Fig. 8 is an ultraviolet-visible absorption spectrum diagram of a mixed solution of ABDA and the aqueous solution of monodentate gold nanoparticles prepared in example 1 placed for different times in a singlet oxygen generation process under a dark condition.
Fig. 9 is an ultraviolet-visible absorption spectrum diagram of a mixed solution of ABDA and the aqueous solution of monodentate gold nanoparticles prepared in example 1 placed for different times in a singlet oxygen generation process under illumination conditions.
FIG. 10 is a diagram showing the UV-VIS absorption spectrum of the 0.1mmol/L aqueous solution of the dual ligand gold nanoparticles prepared in example 1.
Fig. 11 is an ultraviolet-visible absorption spectrum of a mixed solution of ABDA and the aqueous solution of the gold nanoparticles with the double ligands prepared in example 1 placed for different times in a dark condition during the generation of singlet oxygen.
Fig. 12 is an ultraviolet-visible absorption spectrum diagram of a mixed solution of ABDA and the aqueous solution of the gold nanoparticles with double ligands prepared in example 1 placed for different times in the process of generating singlet oxygen under illumination conditions.
FIG. 13 is a graph of the UV-VIS absorption spectra of blank ABDA solutions in example 1 under dark conditions for different periods of time.
FIG. 14 is a graph of the UV-VIS absorption spectra of blank ABDA solutions in example 1 under light conditions for different periods of time.
FIG. 15 is a comparison plot of the linear fit of the single ligand versus the dual ligand gold nanoparticle aqueous solution prepared in example 1 with different illumination times versus the percentage of unreacted ABDA in the initial amount.
FIG. 16 is a graph showing the results of the cell experiment in example 1.
FIG. 17 is a comparison graph of fluorescence spectra of the aqueous solutions of mono-and bi-ligand gold nanoparticles prepared in example 2.
FIG. 18 is a comparison plot of the linear fit of the single ligand versus the dual ligand gold nanoparticle aqueous solution prepared in example 2 for different illumination times versus the percentage of unreacted ABDA in the initial amount.
FIG. 19 is a comparison graph of fluorescence spectra of the aqueous solutions of mono-and bi-ligand gold nanoparticles prepared in example 3.
FIG. 20 is a comparison plot of the linear fit of the single ligand versus the dual ligand gold nanoparticle aqueous solution prepared in example 3 for different illumination times versus the percentage of unreacted ABDA in the initial amount.
Detailed Description
The technical solutions of the present invention are further described in detail below with reference to specific examples and drawings, but the scope and implementation of the present invention are not limited thereto.
In the examples, the fluorescence/phosphorescence/luminescence spectrophotometer (LS-55, PerkinElmer, usa) was used as an apparatus for detecting the change in fluorescence generated from the single ligand gold nanoparticles during synthesis, the ultraviolet-visible absorption spectrometer (UV-1780, shimadzu, japan) was used as an apparatus for comparing the generation rates of the single ligand and double ligand gold nanoparticles, the inductively coupled plasma mass spectrometer (ICP-MS, seimer femtoltechnology) was used for quantifying the concentration of the single ligand gold nanoparticles, and the automatic microplate reader (ELX800, Biotek, usa) was used as an apparatus for detecting cytotoxicity.
Example 1
All glassware is soaked and cleaned by aqua regia in advance, 9.0mL of polyethylene glycol monomethyl ether mercaptan aqueous solution (the concentration is 0.05mol/L, the molecular weight of the polyethylene glycol monomethyl ether mercaptan is 800) and 39.5mL of deionized water are added into a three-neck flask with the specification of 100mL under the condition of room temperature, then 1.5mL of chloroauric acid aqueous solution (the concentration is 0.1mol/L) is added under the conditions of 90 ℃ oil bath and magnetic stirring, then stirring is continued under the condition of the stirring speed of 1500rpm/min until the fluorescence intensity of a reaction system is detected to be strongest by a fluorescence/phosphorescence/luminescence spectrophotometer, the reaction is stopped immediately, a dialysis bag with the molecular weight cutoff of 3kDa is used for dialyzing 3 times in the deionized water solution after cooling to the room temperature, and the room temperature is only needed, and each time is not less than 6 hours, so as to obtain the bright yellow single-ligand gold nanoparticle.
For further subsequent operation, the obtained single ligand gold nanoparticle aqueous solution is concentrated to 500 mu L by centrifugation (4000rpm/min) of an ultrafiltration tube with the molecular weight of 3kDa, the solution is taken out to a 1.5mL centrifuge tube, then 2100g of high-speed centrifugation is carried out to remove large particles at the bottom, and then ICP-MS is used for quantifying to obtain 50mmol/L single ligand gold nanoparticle aqueous solution.
Then, in a three-neck flask with the specification of 100mL, 0.1mL of 50mmol/L aqueous solution of the single-ligand gold nanoparticles is added into 50mL of 0.3mmol/L aqueous solution of mercaptoethylamine for reaction, the mercaptoethylamine is the mercapto small molecular compound B, and the solution is still yellow after being stirred at room temperature for 24 hours. And dialyzing in deionized water solution for 3 times at room temperature, wherein the cut-off molecular weight of the dialysis bag is 3kDa, and the time is not less than 6h, thus obtaining the double-ligand gold nanoparticle aqueous solution.
For further subsequent operation, the obtained double-ligand gold nanoparticle aqueous solution is concentrated to 500 mu L by centrifugation (4000rpm/min) of an ultrafiltration tube with the molecular weight of 3kDa, the solution is taken out to a 1.5mL centrifuge tube, then 2100g of high-speed centrifugation is carried out to remove large particles at the bottom, and then ICP-MS is used for quantifying to obtain the 50mmol/L double-ligand gold nanoparticle aqueous solution.
FIG. 1 is a normalized excitation, emission and absorption spectra of the aqueous solution of monodiganded gold nanoparticles prepared in example 1. From the figure it follows that: the maximum emission wavelength of the gold nanoparticles is 610nm, and the excitation wavelength is 390 nm.
FIG. 2 is a transmission electron microscope photograph of the aqueous solution of monodiganded gold nanoparticles prepared in example 1. As can be seen from the figure: the gold nanoparticles are uniformly dispersed in water, and the particle size of the single-ligand gold nanoparticles is 0.5-3 nm.
FIG. 3 is an X-ray photoelectron spectroscopy (XPS) chart of the aqueous solution of monodiganded gold nanoparticles prepared in example 1. Analyzing by XPS peak separation software, fitting peak areas of monovalent gold elements and zero-valent gold element peak areas, and calculating to obtain: wherein the proportion of the univalent gold element in the total gold element is 44.46%.
FIG. 4 is a comparison chart of fluorescence spectra of the aqueous solution of mono-ligand gold nanoparticles and the aqueous solution of bi-ligand gold nanoparticles prepared in example 1.
FIG. 5 is a TEM image of the aqueous solution of dual ligand gold nanoparticles prepared in example 1. As can be seen from the figure: the double-ligand gold nanoparticles are uniformly dispersed in water, and the particle size of the double-ligand gold nanoparticles is 0.5-3 nm.
Fig. 6 is a Zeta potential diagram of the aqueous solution of mono-ligand gold nanoparticles and di-ligand gold nanoparticles synthesized in example 1 under the condition of pH 7.4. As can be seen from the figure: the single ligand gold nanoparticles are negatively charged, while the double ligand gold nanoparticles are partially positively charged.
In order to detect the rate of singlet oxygen generation of the synthesized aqueous solution of the monodentate gold nanoparticles, a singlet oxygen-specific detector was used: 9, 10-anthracenediyl-bis (methylene) dipropanedioic acid (ABDA for short, > 99%) has four characteristic absorption peaks 342,359,378 and 400nm in an ultraviolet-visible absorption spectrum, and the detection principle is that once singlet oxygen is generated in a solution, the ABDA immediately captures the singlet oxygen in the solution and reacts to generate an endogenous oxidation product, so that the four characteristic absorption peaks of the ABDA are reduced, and the reaction formula is as follows:
wherein the rate of decrease of the ABDA absorption peak corresponds to the rate of production of singlet oxygen. And testing the change of the ultraviolet visible absorption spectrum of the sample to be tested and the ABDA mixed solution under different illumination time by using an ultraviolet visible spectrophotometer to obtain the rate of generating the singlet oxygen.
The method for detecting the singlet oxygen generation rate of the single-ligand gold nanoparticles comprises the following steps: the specific experimental steps are that after an ultraviolet visible absorption spectrometer is used and an instrument is preheated for 15min, 500 mu L of 0.04mol/L BR buffer solution (pH 7.4) is respectively added into two 700 mu L cuvettes, one serves as a sample cell, the other serves as a reference cell, a base line (300-600 nm) is firstly swept, then a blank is swept, the blank is guaranteed to fluctuate within +/-0.0005 at the position of 300-600 nm, the sample cell is clean, then 1 mu L of quantitative 50mmol/L of single-ligand gold nanoparticle aqueous solution is added into the sample cell, the absorption spectrum (the concentration is 0.1mmol/L) of the single-ligand gold nanoparticle aqueous solution is measured (the test result is shown in figure 7), then 10 mu L of prepared 10mmol/L ABDA solution is added, the mixture is uniform, the absorption spectrum is measured every 5min under the condition of light shielding, the total 15min, then the DMSO is taken out and placed into a 1.5mL centrifuge tube, using a laser (450nm 100 mW/cm)2) The mixed solution of the single ligand gold nanoparticles and the ABDA is illuminated, the absorption spectrum is measured once every 5min of illumination, the illumination time is kept for 15min, and the test result is shown in figure 8.
FIG. 7 is the UV-VIS absorption spectrum of 0.1mmol/L aqueous solution of single ligand gold nanoparticles.
Fig. 8 is an ultraviolet-visible absorption spectrum diagram of a mixed solution of the singlet oxygen indicator ABDA and the aqueous solution of the monodentate gold nanoparticles prepared in example 1 placed for different times under a dark condition in the process of singlet oxygen generation. As can be seen from the figure: the single ligand gold nano particles are placed for 5min, 10min and 15min to coincide with the line of 0min, which shows that the single ligand gold nano particles do not generate singlet oxygen under the condition of keeping out of the sun.
Fig. 9 is an ultraviolet-visible absorption spectrum diagram of a mixed solution of the singlet oxygen indicator ABDA and the aqueous solution of the monodentate gold nanoparticles prepared in example 1 in the singlet oxygen generation process placed for different periods of time under illumination. As can be seen from the figure: with the increase of the illumination time, the absorption of the mixed solution at 380nm gradually decreases, which shows that the single ligand gold nanoparticles generate singlet oxygen under the illumination condition.
The method for detecting the rate of singlet oxygen generated by the double-ligand gold nanoparticles comprises the following steps: the method for detecting the rate of the singlet oxygen generated by the double-ligand gold nanoparticles is the same as the method for detecting the rate of the singlet oxygen generated by the single-ligand gold nanoparticles.
FIG. 10 is a UV-VIS absorption spectrum of a 0.1mmol/L aqueous solution of dual ligand gold nanoparticles.
Fig. 11 is an ultraviolet-visible absorption spectrum of a mixed solution of ABDA and the aqueous solution of the gold nanoparticles with the double ligands prepared in example 1 placed for different times in a dark condition during the generation of singlet oxygen. As can be seen from the figure: the placing for 5min, 10min and 15min is overlapped with the 0min line, which shows that the double-ligand gold nano particles do not generate singlet oxygen under the condition of keeping out of the sun.
Fig. 12 is an ultraviolet-visible absorption spectrum diagram of a mixed solution of the singlet oxygen indicator ABDA and the aqueous solution of the dual-ligand gold nanoparticles prepared in example 1 in the process of generating singlet oxygen, placed for different times under illumination conditions. As can be seen from the figure: with the increase of the illumination time, the absorption of the mixed solution at 380nm gradually decreases, which shows that the double-ligand gold nanoparticles generate singlet oxygen under the illumination condition.
Blank group: in order to eliminate the influence of ABDA degradation and absorption peak reduction caused by photodegradation, a group of blank groups is set, 1 mu L of deionized water is added into the blank groups, and other operations are carried out in the same way as the method for detecting the rate of singlet oxygen generation of the single-ligand gold nanoparticles.
FIG. 13 is a graph of the UV-VIS absorption spectra of blank ABDA solutions after exposure to light. As can be seen from the figure: the ABDA was stable under dark conditions when placed for 5min, 10min, 15min and 0min coincident.
FIG. 14 is a graph of the UV-VIS absorption spectra of a blank set of ABDA solutions after exposure to light for various periods of time. As can be seen from the figure: placing for 5min, 10min and 15min to coincide with the 0min line, which shows that the ABDA is kept stable under the illumination condition, and further shows that the absorption peak of the ABDA is reduced because the single-ligand gold nanoparticles and the double-ligand gold nanoparticles generate singlet oxygen through illumination.
Calculating the enhancement efficiency of the double-ligand gold nanoparticles for generating singlet oxygen: because the concentrations of the two types of nanoparticles are the same when the singlet oxygen generation rate is detected, the enhancement efficiency of the double-ligand gold nanoparticles is as follows: the ratio of the ABDA decrease slope is caused by the increase of the double-ligand gold nanoparticles and the single-ligand gold nanoparticles along with the illumination time. Firstly, calculating the percentage of the unreacted ABDA in the mixed solution of the single-ligand gold nanoparticle aqueous solution and the ABDA at different times under illumination to the initial amount, respectively taking the absorption value at 380nm on each spectrogram in FIG. 9, deducting the absorption of 0.1mmol/L of the single-ligand gold nanoparticles at 380nm (as shown in FIG. 7), obtaining the value representing the unreacted ABDA at different time points under illumination, then respectively dividing the value by the illumination for 0min, and multiplying the value by 100%, thus obtaining the percentage of the unreacted ABDA in the mixed solution of the single-ligand gold nanoparticle aqueous solution and the ABDA at different times under illumination to the initial amount. And then calculating the percentage of the unreacted ABDA in the double-ligand gold nanoparticle aqueous solution and the ABDA mixed solution in different times of illumination, respectively taking the absorption value at 380nm on each spectrogram in the graph 12, deducting the absorption of 0.1mmol/L double-ligand gold nanoparticles at 380nm (shown in the graph 10), obtaining the value representing the unreacted ABDA in the double-ligand gold nanoparticle aqueous solution and the ABDA mixed solution at different time points of illumination, respectively dividing the value by the illumination for 0min, and multiplying the value by 100%, thereby obtaining the percentage of the unreacted ABDA in the double-ligand gold nanoparticle aqueous solution and the ABDA mixed solution in different times of illumination. The time points (0, 5, 10, and 15) of the illumination were plotted as abscissa and the percentage of the unreacted ABDA in the initial amount in the illumination was plotted as ordinate, and the graph was obtained by linear fitting as fig. 15.
FIG. 15 is a comparison of the linear fit of the single ligand versus double ligand gold nanoparticle aqueous solutions prepared in example 1 with different illumination times versus the percentage of unreacted ABDA relative to the starting amount. From the straight line fit in fig. 15, it can be seen that: the ABDA amount decreasing slope is-4.40 due to the increase of the single-ligand gold nanoparticles along with the illumination time, the ABDA decreasing slope is-8.24 due to the increase of the double-ligand gold nanoparticles along with the illumination time, and the calculation is finally carried out to obtain the following components: the rate of singlet oxygen production by the dual ligand gold nanoparticles was enhanced by a percentage (-8.24- (-4.40))/(-4.40)) × 100% ═ 87% compared to the single ligand gold nanoparticles.
Cell experiments
HeLa cells (HeLa cell line supplied by southern medical university, Guangzhou) were used as a model in vitro, and 18 wells in the middle were taken 12 hours in advance in two 96-well plates, each cultured at 1X 104Each well of HeLa cells was supplemented with 100 μ L of newly prepared medium containing 10% (v/v) south america, uray super fetal bovine serum, 1% (v/v) penicillin-streptomycin (yiwei jie (shanghai) trade ltd) high-glucose medium (DMEM), one 96-well plate as light-protected group, and one as light-protected group. After 12 hours, 0.4. mu.L of 50mmol/L aqueous solution of the monodiganded gold nanoparticles prepared in example 1 was added per well to 6 wells of a 96-well plate of the light group, 0.4. mu.L of 50mmol/L aqueous solution of the bidentate gold nanoparticles prepared in example 1 was added per well to another 6 wells as a blank, and 0.4. mu.L deionized water was added per well, representing 6 parallel experiments. The light-shielded 96-well plate was operated in the same manner as the light-shielded 96-well plate. Then, both 96-well plates were placed in an incubator (37 ℃ C., 5% CO)2) Culturing for 6h, taking out 96-well plate of the illumination group, and illuminating with blue LED lamp at room temperature (wavelength of 450nm and optical density of 2.5 mW/cm)2)4h, placing the 96 pore plate of the light-proof group in a room temperature light-proof culture mode for 4h, then continuously placing the 96 pore plate of the illumination group and the light-proof group in an incubator for culture for 14h, then adding 20 mu L of 5mg/mL MTT solution prepared by phosphate buffer solution into each pore, continuously placing the 96 pore plate of the illumination group and the light-proof group in the incubator for culture for 4h, then taking all the solution in the pore away, immediately adding 150 mu L of DMSO solution into each pore, oscillating by using a 96 pore plate oscillator for 10min 1000rpm/min, and then testing the absorption value of 490nm by using an automatic microplate reader, namely, the number of the cells corresponding to survival. The average absorbance of 6 replicates was calculated, divided by the average absorbance of the blank in the dark group, and multiplied by 100% to represent the cell viability (%), and the results are shown in FIG. 16.
FIG. 16 is a graph showing the results of the cell experiment in example 1. As can be seen from the figure: under the condition of keeping out of the sun, both the two kinds of gold nanoparticles have no toxicity to cells, and under the condition of illumination, the toxicity of the double-ligand gold nanoparticles to the cells is obviously higher than that of the single-ligand gold nanoparticles, and the illumination group of the blank group has almost no toxicity to the cells.
Example 2
All glassware is soaked and cleaned by aqua regia in advance, 12.0mL of sulfhydryl polyethylene glycol amino aqueous solution (with the concentration of 0.1mol/L and the molecular weight of sulfhydryl polyethylene glycol amino is 1000) and 36.5mL of deionized water are added into a three-neck flask with the specification of 100mL under the condition of room temperature, then 1.5mL of chloroauric acid aqueous solution (with the concentration of 0.1mol/L) is added under the conditions of 95 ℃ oil bath and magnetic stirring, then stirring is continued under the condition of the stirring speed of 1500rpm/min until the fluorescence intensity of a reaction system detected by a fluorescence/phosphorescence/luminescence spectrophotometer reaches the maximum intensity, the reaction is stopped immediately, a dialysis bag with the molecular weight cutoff of 3kDa is used for dialysis 3 times in the deionized water solution after cooling to the room temperature, and the room temperature is not less than 6 hours each time, so as to obtain the bright yellow gold single-ligand nano particle aqueous solution.
For further subsequent operation, the obtained single ligand gold nanoparticle aqueous solution is concentrated to 500 mu L by centrifugation (4000rpm/min) of an ultrafiltration tube with the molecular weight of 3kDa, the solution is taken out to a 1.5mL centrifuge tube, then 2100g of high-speed centrifugation is carried out to remove large particles at the bottom, and then ICP-MS is used for quantifying to obtain 50mmol/L single ligand gold nanoparticle aqueous solution.
Then, in a three-neck flask with the specification of 100mL, 0.1mL of 50mmol/L aqueous solution of the single-ligand gold nanoparticles is added into 50mL of 0.3mmol/L aqueous solution of mercaptoethylamine for reaction, the mercaptoethylamine is the mercapto small molecular compound B, and the solution is still yellow after being stirred at room temperature for 24 hours. And dialyzing in deionized water solution for 3 times at room temperature, wherein the cut-off molecular weight of the dialysis bag is 3kDa, and the time is not less than 6h, thus obtaining the double-ligand gold nanoparticle aqueous solution.
For further subsequent operation, the obtained double-ligand gold nanoparticle aqueous solution is concentrated to 500 mu L by centrifugation (4000rpm/min) of an ultrafiltration tube with the molecular weight of 3kDa, the solution is taken out to a 1.5mL centrifuge tube, then 2100g of high-speed centrifugation is carried out to remove large particles at the bottom, and then ICP-MS is used for quantifying to obtain the 50mmol/L double-ligand gold nanoparticle aqueous solution.
FIG. 17 is a comparison graph of fluorescence spectra of the aqueous solutions of mono-and bi-ligand gold nanoparticles prepared in example 2.
The method for detecting the rate of singlet oxygen generation of the monodentate and bidentate gold nanoparticles synthesized in example 2 and the method for calculating the enhanced efficiency of singlet oxygen generation of the bidentate gold nanoparticles are the same as those of example 1.
FIG. 18 is a comparison plot of the linear fit of the single ligand versus the dual ligand aqueous gold nanoparticle solutions synthesized in example 2 with different illumination times versus the percentage of unreacted ABDA in the starting amount. From the straight line fit in fig. 18, it can be seen that: the ABDA decreasing slope is-3.04 due to the increase of the single-ligand gold nanoparticle aqueous solution along with the illumination time, the ABDA decreasing slope is-8.24 due to the increase of the double-ligand gold nanoparticle aqueous solution along with the illumination time, and the final calculation results are as follows: the rate of singlet oxygen production from aqueous solutions of dual ligand gold nanoparticles was enhanced by a percentage of (-8.24- (-3.04))/(-3.04)) x 100% (-171% compared to aqueous solutions of single ligand gold nanoparticles.
Example 3
All glassware is soaked and cleaned by aqua regia in advance, 4.8mL of sulfhydryl polyethylene glycol carboxyl aqueous solution (the concentration is 0.05mol/L, and the molecular weight of sulfhydryl polyethylene glycol carboxyl is 1000) and 14.6mL of deionized water are added into a three-neck flask with the specification of 50mL under the condition of room temperature, then 0.6mL of chloroauric acid aqueous solution (the concentration is 0.1mol/L) is added under the conditions of 95 ℃ oil bath and magnetic stirring, then stirring is continued under the condition of stirring speed of 1500rpm/min until the fluorescence intensity of a reaction system detected by a fluorescence/phosphorescence/luminescence spectrophotometer reaches the maximum intensity, the reaction is stopped immediately, a dialysis bag with the molecular weight cutoff of 3kDa is used for dialysis 3 times in the deionized water solution after cooling to the room temperature, and the room temperature is not less than 6 hours each time, so as to obtain the bright yellow gold single ligand nano particle aqueous solution.
For further subsequent operation, the obtained single ligand gold nanoparticle aqueous solution is concentrated to 500 mu L by centrifugation (4000rpm/min) of an ultrafiltration tube with the molecular weight of 3kDa, the solution is taken out to a 1.5mL centrifuge tube, then 2100g of high-speed centrifugation is carried out to remove large particles at the bottom, and then ICP-MS is used for quantifying to obtain 50mmol/L single ligand gold nanoparticle aqueous solution.
Then, in a three-neck flask with the specification of 100mL, 0.1mL of 50mmol/L aqueous solution of the single-ligand gold nanoparticles is added into 50mL of 0.3mmol/L aqueous solution of mercaptoethylamine for reaction, the mercaptoethylamine is the mercapto small molecular compound B, and the solution is still yellow after being stirred at room temperature for 24 hours. And dialyzing in deionized water solution for 3 times at room temperature, wherein the cut-off molecular weight of the dialysis bag is 3kDa, and the time is not less than 6h, thus obtaining the double-ligand gold nanoparticle aqueous solution.
For further subsequent operation, the obtained double-ligand gold nanoparticle aqueous solution is concentrated to 500 mu L by centrifugation (4000rpm/min) of an ultrafiltration tube with the molecular weight of 3kDa, the solution is taken out to a 1.5mL centrifuge tube, then 2100g of high-speed centrifugation is carried out to remove large particles at the bottom, and then ICP-MS is used for quantifying to obtain the 50mmol/L double-ligand gold nanoparticle aqueous solution.
FIG. 19 is a comparison graph of fluorescence spectra of the aqueous solutions of mono-and bi-ligand gold nanoparticles prepared in example 3.
The method for detecting the rate of singlet oxygen generated by the aqueous solution of the single-ligand gold nanoparticles and the double-ligand gold nanoparticles synthesized in the embodiment 3 and the method for calculating the enhancement efficiency of the singlet oxygen generated by the double-ligand gold nanoparticles are the same as the method in the embodiment 1.
FIG. 20 is a comparison plot of the linear fit of the single ligand versus double ligand gold nanoparticle aqueous solutions synthesized in example 3 for different illumination times versus the percentage of unreacted ABDA relative to the starting amount. From the straight line fit in fig. 20, it can be seen that: the ABDA decreasing slope is-1.54 due to the increase of the single-ligand gold nanoparticle aqueous solution along with the illumination time, the ABDA decreasing slope is-4.63 due to the increase of the double-ligand gold nanoparticle aqueous solution along with the illumination time, and the final calculation results are as follows: the rate of enhancement of singlet oxygen generation by the aqueous solution of dual ligand gold nanoparticles compared to the aqueous solution of single ligand gold nanoparticles was (-4.63- (-1.54))/(-1.54)) × 100% ═ 200%.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. The preparation method of the dual-ligand gold nanoparticle aqueous solution is characterized by comprising the following steps of:
(1) adding the sulfydryl micromolecule compound A into water to prepare a sulfydryl micromolecule compound A water solution;
(2) adding a hydrosulphonyl micromolecule compound A aqueous solution into a solvent, adding a chloroauric acid aqueous solution under the conditions of oil bath and stirring, continuously stirring for reaction, stopping the reaction when the fluorescence intensity of a reaction system reaches the strongest value and is not increased any more, cooling to room temperature, putting into a dialysis bag, dialyzing in water, and obtaining a single-ligand gold nanoparticle aqueous solution after the dialysis is finished;
(3) adding the single-ligand gold nanoparticle aqueous solution prepared in the step (2) into a mercapto small molecular compound B aqueous solution, stirring for reaction, putting the mixture into a dialysis bag after the reaction is finished, dialyzing the mixture in water, and obtaining a double-ligand gold nanoparticle aqueous solution with partial positive charges after the dialysis is finished;
the mercapto small molecular compound A in the step (1) has the following structure: R-SH, wherein R is alkyl or heterocycle containing nitrogen or oxygen atom;
the mercapto small molecular compound B in the step (3) is one of mercaptoethylamine, mercaptobenzimidazole and mercaptoimidazole;
the molar ratio of the mercapto small molecular compound A to the chloroauric acid in the step (2) is 1: 1-8: 1;
the molar ratio of the mercapto small molecular compound B to the gold atoms in the single-ligand gold nanoparticle aqueous solution in the step (3) is 0.1: 1-10: 1.
2. the dual-ligand gold nanoparticle aqueous solution according to claim 1, wherein the concentration of the thiol small-molecule compound A in the step (1) is 0.01-0.20 mol/L;
the concentration of the hydrosoluble solution of the mercapto small molecular compound B in the step (3) is 0.01 mmol/L-1 mmol/L.
3. The aqueous solution of dual ligand gold nanoparticles as claimed in any one of claims 1 to 2, wherein the final concentration of the chloroauric acid in the reaction system in step (2) is 1 to 5 mmol/L.
4. The dual-ligand gold nanoparticle aqueous solution according to any one of claims 1 to 2, wherein the final concentration of gold atoms in the single-ligand gold nanoparticle aqueous solution in the step (3) in the reaction system is 0.08mmol/L to 20 mmol/L.
5. The dual ligand gold nanoparticle aqueous solution according to any one of claims 1-2, wherein the dialysis bags of step (2) and step (3) have a molecular weight cut-off of 3-10 kDa;
the dialysis in the step (2) and the step (3) is specifically as follows: dialyzing for at least three times, wherein each time is not less than 6 h.
6. The dual-ligand gold nanoparticle aqueous solution according to any one of claims 1-2, wherein the sulfhydryl small-molecule compound A in the step (1) is one of polyethylene glycol monomethyl ether thiol, sulfhydryl polyethylene glycol amino, sulfhydryl polyethylene glycol carboxyl, glutathione, bovine serum albumin and mercaptopropionic acid;
the solvent in the step (2) is one of ethanol, methanol, tetrahydrofuran, acetonitrile and water.
7. The dual ligand gold nanoparticle aqueous solution according to claim 1, wherein the temperature of the oil bath in the step (2) is 25-95 ℃;
the speed of the continuous stirring in the step (2) is 1000 rpm/min-1500 rpm/min;
and (4) stirring for 10 min-48 h.
8. Use of an aqueous solution of biligand gold nanoparticles as claimed in any one of claims 1 to 7 for the preparation of a photosensitizer.
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