Nanoparticle, preparation and application thereof, and intracellular adriamycin concentration detection sensor

Abstract

The invention discloses a nanoparticle, preparation and application thereof and an intracellular adriamycin concentration detection sensor. The nano particles comprise nano carrier particles, the surfaces of the nano carrier particles are coupled with gold nano-clusters, and the surfaces of the gold nano-clusters and / or the nano carrier particles are coupled with iRGD peptides. The preparation method comprises the following steps: dispersing nano-carrier particles coupled with the gold nano-clusters in a buffer solution containing a coupling agent, activating, adding the iRGD peptide, stirring for reaction, filtering, washing and precipitating to obtain the iRGD-modified composite nano-particles. The nanoparticle can be applied to electrochemical detection of concentration of adriamycin in cells or fluorescence imaging of living cells. The nanoparticles have dual effects of targeting and membrane permeation, not only can target fixation of breast cancer cells on the surface of the modified gold electrode be realized, but also more adriamycin is allowed to enter the cells, and electrochemical signals are transmitted out of the cells in a transmembrane manner under the action of DNA (deoxyribonucleic acid); meanwhile, the nanoparticles have a cell imaging function, so that the cell uptake condition can be conveniently and intuitively observed.

Description

Technical Field

[0001] This invention relates to a technology for detecting intracellular antibiotic concentration, and more particularly to a nanoparticle, its preparation, application, and a sensor for detecting intracellular doxorubicin concentration. Background Technology

[0002] Doxorubicin is an anthracycline antibiotic with potent antitumor activity, widely used clinically in the treatment of cancers such as breast cancer, ovarian cancer, and leukemia. As a chemotherapy drug, a single intravenous infusion of doxorubicin results in a concentration of 60-75 mg / m², primarily exerting its antitumor effect by interfering with intracellular DNA and RNA synthesis. Its killing effect on tumor cells depends on the intracellular drug concentration. Therefore, measuring the intracellular concentration of doxorubicin in tumor cells provides crucial information for tumor diagnosis and treatment, disease progression, and patient quality of life.

[0003] Electrochemical analysis is a detection technique that converts physiological activities within cells into electrical signals. Due to its simplicity, convenience, speed, and sensitivity, it is widely used for the detection of small and large biological molecules. For example, Chinese patent CN115901892A discloses a method for quantitative / qualitative detection of antibiotics in solution using DNA as a recognition element. While this technique can detect the concentration of antibiotics such as oxytetracycline in the target solution, it requires the antibiotics to be uniformly dispersed in the target solution and able to freely bind to the DNA recognition element. When the target is changed from solution to living cells, as the antibiotics enter the cells, they are blocked by the cell membrane, preventing them from freely binding to the extracellular DNA recognition element. Furthermore, the concentration of antibiotics differs between the solution and the cells in the cell suspension, resulting in uneven dispersion. Therefore, when the above method is used to detect the concentration of antibiotics within living cells, the obtained electrochemical signal is difficult to establish an accurate linear relationship with the intracellular antibiotic concentration, thus making it impossible to quantitatively detect the concentration of antibiotics within living cells. It is evident that existing electrochemical detection technologies cannot cross cell membranes to identify and accurately acquire the electrochemical signals of antibiotics within living cells. Therefore, how to use electrochemical detection technologies to quantitatively detect the concentration of doxorubicin within cells has become an urgent technical problem to be solved.

[0004] RGD transmembrane peptide is a short peptide composed of three amino acids: arginine, glycine, and aspartic acid. It is widely distributed in organisms and can specifically bind to integrins αvβ3 and αvβ5, which are overexpressed in tumor cells, mediating various pathophysiological processes. The iRGD peptide is cyclic and possesses both the RGD sequence and a C-terminal sequence (Cend R), exhibiting both targeting and transmembrane permeation functions. Summary of the Invention

[0005] Objectives of this invention: The objective of this invention is to provide nanoparticles for electrochemical detection of doxorubicin concentration in living cells. A second objective is to provide a method for preparing nanoparticles, addressing the problem of how to prepare nanoparticles modified with iRGD peptides. A third objective is to provide an application of these nanoparticles in the electrochemical detection of doxorubicin concentration in cells or in live-cell fluorescence imaging, addressing the problem of how to electrochemically detect doxorubicin concentration in living cells or perform live-cell fluorescence imaging. A fourth objective is to provide a detection sensor for detecting doxorubicin concentration in living cells.

[0006] Technical solution: The present invention provides a nanoparticle comprising a nanocarrier particle, wherein gold nanoclusters are coupled to the surface of the nanocarrier particle, and the gold nanoclusters and / or the nanocarrier particle are coupled to the surface of an iRGD peptide (amino acid sequence CRGDKGPDC) with an amino acid sequence as shown in SEQ ID No. 1.

[0007] In this invention, nanoparticles surface-modified with iRGD peptides can act as "keys" to open cell membranes, facilitating the binding of DNA molecules on the modified electrode surface to intracellular doxorubicin. This enables targeted immobilization of cells on the modified electrode surface and transmembrane transduction of the electrochemical signal of doxorubicin, allowing for quantitative detection of intracellular doxorubicin concentration. Modifying iRGD onto fluorescent composite nanoparticles not only increases the tropism and targeting of nanoparticles to tumor cells but also enhances membrane permeability and enables cell imaging.

[0008] Preferably, in step (1), the nanocarrier particles include at least one of SiO2 nanoparticles and gold nanoparticles, the particle size of the nanocarrier particles does not exceed 300 nm, and the particle size of the gold nanoclusters does not exceed 15 nm.

[0009] A second aspect of this invention discloses a method for preparing the above-mentioned nanoparticles, comprising the following steps: The nanocarrier particles coupled with gold nanoclusters were dispersed in a buffer solution containing a coupling agent, activated, and then iRGD peptides were added. After stirring and reacting, the precipitate was filtered and washed to obtain iRGD-modified composite nanoparticles.

[0010] Preferably, the method for preparing the nanocarrier particles coupled with gold nanoclusters is as follows: Amino-modified SiO2 nanoparticles were dispersed into gold nanoclusters, and then N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were added for activation. The reaction was continued with stirring, and after centrifugation and washing, silica / gold nanoclusters (SiO2 / AuNCs) composite fluorescent nanoparticles were obtained.

[0011] In some embodiments, the ratio of amino-modified SiO2 nanoparticles to gold nanoclusters is 10-30 mg: 1-3 mL; In some embodiments, bovine serum albumin (BSA) aqueous solution and chloroauric acid aqueous solution are placed in a reaction flask, and NaOH aqueous solution is added under vigorous stirring. Then, the reaction is carried out in a water bath at 37 °C in the dark with stirring. The solution color changes from bright yellow to dark brown, and gold nanoclusters are obtained.

[0012] Preferably, the coupling agent is N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the mass ratio of the gold nanocluster-coupled nanocarrier particles to the iRGD peptide is 3-7:1; the stirring reaction conditions are 100-200 rpm, room temperature, and light-protected stirring reaction for 12-48 h.

[0013] The third aspect of this invention discloses the application of the above-mentioned nanoparticles in the electrochemical detection of intracellular doxorubicin concentration or in live-cell fluorescence imaging.

[0014] Preferably, the above application method specifically includes the following steps: (1) Add the nanoparticles and doxorubicin at different final concentrations to the live cell suspension, incubate, centrifuge, and wash to remove unbound nanoparticles and doxorubicin to obtain the cells to be tested; (2) The cells to be tested are resuspended in culture medium to obtain a cell suspension, and the cell suspension is subjected to laser confocal imaging; Alternatively, a gold electrode with the DNA sequence shown in SEQ ID No. 2 (TGG ATG ATG TGG TAT GGA AC) can be inserted into the cell suspension to be tested. After incubation, the gold electrode can be transferred to a buffer solution for electrochemical detection. A linear relationship can be established between the measured electrochemical signal and the intracellular doxorubicin concentration after treatment with different final concentrations of doxorubicin. The intracellular doxorubicin concentration can be calculated based on this linear relationship and the electrochemical signal of the cell sample to be tested.

[0015] Preferably, in step (1), the final concentration of doxorubicin is in the range of 0.05-200 μM, the molar ratio of iRGD peptide on the surface of nanoparticles to doxorubicin is 1-20:5, and the incubation conditions are 36-38℃ for 1-4 h. In step (2), the electrochemical signal is the current intensity measured by differential pulse voltammetry (DPV), the incubation conditions are 36-38℃ for 3-9h; the buffer is Tris-HCl buffer. The cells in question are tumor cells.

[0016] In some embodiments, the concentration of the live cell suspension in step (1) is 1×10⁻⁶. 5 -1×10 7The cell / mL concentration was 4-8:1, and the volume ratio of live cell suspension to doxorubicin solution was 4-8:1; the concentration of the cell suspension to be tested in step (2) was 5×10⁻⁶. 5 -5×10 6 cell / mL.

[0017] Preferably, the method for preparing the gold electrode with the DNA sequence modified on its surface is as follows: The gold electrode was immersed in a HAuCl4 solution containing KNO3 and electrodeposited at -0.1 to -0.3 V for 10-30 s to obtain AuNPs / Au electrodes; AuNPs / Au electrodes were incubated in 0.5–1.5 μM SH-DNA solution for 8–24 h to obtain gold electrodes with surface-modified DNA sequences via Au-S bond binding. SH-DNA is thiol-modified DNA with the sequence TGG ATG ATG TGG TAT GGAAC-(CH2)6-SH.

[0018] A fourth aspect of this invention discloses an electrochemical sensor for detecting intracellular doxorubicin concentration, comprising a gold electrode with a surface modified with the DNA sequence shown in SEQ ID No. 2, a reference electrode, a counter electrode, an incubation tank containing an incubation solution, and an electrochemical detection tank containing a buffer solution, wherein the incubation solution contains the aforementioned nanoparticles. The reference electrode and counter electrode are inserted into the electrochemical detection tank, and the gold electrode with the surface modified with the DNA sequence shown in SEQ ID No. 2 can be selectively inserted into either the incubation tank or the electrochemical detection tank according to the operating steps. The gold electrode, reference electrode, and counter electrode are all electrically connected to an external electrochemical measuring device when detecting an electrical signal.

[0019] In some embodiments, the reference electrode may be a saturated calomel electrode or an Ag / AgCl electrode; the counter electrode may be a platinum electrode.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) SiO2 / AuNCs-iRGD nanoparticles have both targeting and permeabilizing effects. They can achieve targeted immobilization of breast cancer cells on the modified gold electrode surface, and allow more doxorubicin to enter the cells. Through the action of DNA, electrochemical signals are transmitted across the membrane out of the cells, thereby improving the sensitivity of detection. (2) SiO2 / AuNCs-iRGD can emit red fluorescence and has cell imaging function, which makes it convenient to observe cell uptake directly; (3) After modifying AuNPs by electrodeposition, the specific surface area of ​​the gold electrode is significantly increased, which can modify more DNA molecules, thereby binding more doxorubicin and further improving the detection sensitivity.

[0021] (4) Electrochemical detection methods are simple, convenient, fast and sensitive, and can achieve trace detection of target substances. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the preparation process of SiO2 / AuNCs-iRGD nanoparticles and the electrochemical detection principle of intracellular doxorubicin concentration in this invention. Figure 2 Characterization diagram of SiO2 / AuNCs-iRGD nanoparticles prepared in this invention; Figure 3 This is a characterization diagram of the DNA-modified gold electrode in this invention; Figure 4 This is an AC impedance characterization diagram of the assembly process of breast cancer cells on the electrode surface in this invention; Figure 5 This is a graph showing the results of the laser confocal imaging performance of SiO2 / AuNCs-iRGD nanoparticles in this invention; Figure 6 This is an optimized diagram showing the concentration of SiO2 / AuNCs-iRGD nanoparticles added in this invention; Figure 7 The graph shows the linear relationship between the current intensity measured by DPV and the concentration of doxorubicin in breast cancer cells. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0024] Example 1: A method for preparing nanoparticles is as follows: 5.0 mL of 5% bovine serum albumin (BSA) aqueous solution and 5.0 mL of chloroauric acid aqueous solution (HAuCl4, 10.0 mM) were placed in a reaction flask, and 0.5 mL of NaOH aqueous solution (1.0 M) was added under vigorous stirring. The reaction was then carried out in a water bath at 37 °C in the dark for 12 h with stirring. The solution color changed from bright yellow to dark brown, yielding gold nanoclusters (AuNCs).

[0025] The preparation method of silica nanospheres (SiO2NSs) is as follows: 90 mL of 95% ethanol, 2.7 mL of ammonia, and 5 mL of double-distilled water were sequentially added to a 100 mL beaker after treatment. Then, 2.7 mL of tetraethyl orthosilicate (TEOS) was added, and the mixture was sonicated for 10 min to ensure thorough mixing. The mixture was then magnetically stirred for 24 h. The resulting mixture was centrifuged at 10000 r / min for 15 min, and the precipitate was washed three times with distilled water and dried in an oven to obtain a solid powder, thus yielding SiO2NSs.

[0026] The amino-functionalization method for silica nanospheres is as follows: Accurately weigh 100 mg SiO2NSs, ultrasonically disperse it in 10 mL of anhydrous ethanol, add 0.4 mL of 3-aminopropyltriethoxysilane (APTES) and mix well. After stirring magnetically for 6 h, centrifuge the suspension, wash the precipitate three times with distilled water, and dry it in an oven to obtain amino-functionalized silica nanospheres (NH2-SiO2NSs).

[0027] 20 mg of NH2-SiO2NSs was dispersed in 2.0 mL of AuNCs, followed by the addition of 2 mg NHS and 2 mg EDC for activation for 30 min. The reaction was then continued with magnetic stirring at room temperature for 12 h. During the reaction, the carboxyl groups on the AuNCs surface underwent amidation with the amino groups on the SiO2NSs surface, thus assembling AuNCs onto the SiO2NSs surface. After centrifugation and washing with water, silica / gold nanoclusters (SiO2 / AuNCs) composite fluorescent nanoparticles were obtained. 20 mg of SiO2 / AuNCs composite fluorescent nanoparticles were ultrasonically dispersed in 2 mL of PBS buffer solution (pH 7.0), and then an appropriate amount of crosslinking agent (2 mg NHS and 2 mg EDC) was added. The mixture was then activated with stirring at room temperature for 30 min. 4 mg of iRGD peptide (amino acid sequence CRGDKGPDC) was added to the mixture, and the reaction was stirred at room temperature for 24 h. After centrifugation and discarding the supernatant, the precipitate was washed three times with PBS to obtain SiO2 / AuNCs-iRGD nanoparticles.

[0028] The morphology of SiO2 / AuNCs-iRGD nanoparticles and related particles was examined by electron microscopy, and the results are as follows: Figure 2 As shown, Figure 2 Image A in the middle is a high-magnification transmission electron microscope image of AuNCs; Figure 2 Image B in the middle is a transmission electron microscope image of SiO2 nanoparticles; Figure 2 Image C in the middle is a transmission electron microscope image of SiO2 / AuNCs; Figure 2 Image D in the middle is a transmission electron microscope image of SiO2 / AuNCs-iRGD; Figure 2 The middle image (E) shows the fluorescence spectrum of SiO2 / AuNCs-iRGD.

[0029] like Figure 2 As shown in Figures A and E, the synthesized AuNCs have a particle size of approximately 5 nm. Their solution exhibits red fluorescence under UV light irradiation, with a maximum emission wavelength of 650 nm. AuNCs were deposited onto SiO2 nanoparticles (such as...). Figure 2 As shown in Figure C, after coupling iRGD peptides onto SiO2 / AuNCs (with a particle size of approximately 140 nm), as shown... Figure 2As shown in Figure D, many small black dots can be seen deposited on the surface of SiO2 nanoparticles, with a particle size of about 160 nm, which can also emit red fluorescence.

[0030] Example 2: A method for preparing nanoparticles is as follows: Following the method in Example 1, 20 mg of NH2-SiO2NSs was dispersed in 2.0 mL of AuNCs, followed by the addition of 2 mg NHS and 2 mg EDC for activation for 30 min. The mixture was then magnetically stirred at room temperature for 12 h. After centrifugation and washing, silica / gold nanoclusters (SiO2 / AuNCs) composite fluorescent nanoparticles were obtained. 24 mg of SiO2 / AuNCs composite fluorescent nanoparticles were ultrasonically dispersed in 2 mL of PBS buffer solution (pH 7.0), and then an appropriate amount of crosslinking agent (2 mg NHS and 2 mg EDC) was added. The mixture was then activated by stirring at room temperature for 40 min. 8 mg of iRGD peptide was added to the above mixture, and the reaction was stirred at room temperature for 12 h. After centrifugation and discarding the supernatant, the precipitate was washed three times with PBS to obtain SiO2 / AuNCs-iRGD nanoparticles.

[0031] Example 3: A method for preparing nanoparticles is as follows: Following the method in Example 1, 20 mg of NH2-SiO2NSs was dispersed in 2.0 mL of AuNCs, followed by the addition of 2 mg NHS and 2 mg EDC for activation for 30 min. The mixture was then magnetically stirred at room temperature for 12 h. After centrifugation and washing, silica / gold nanoclusters (SiO2 / AuNCs) composite fluorescent nanoparticles were obtained. 14 mg of SiO2 / AuNCs composite fluorescent nanoparticles were ultrasonically dispersed in 2 mL of PBS buffer solution (pH 7.0), and then an appropriate amount of crosslinking agent (2 mg NHS and 2 mg EDC) was added. The mixture was then activated by stirring at room temperature for 20 min. 2 mg of iRGD peptide was added to the mixture, and the reaction was stirred at room temperature for 48 h. After centrifugation and discarding the supernatant, the precipitate was washed three times with PBS to obtain SiO2 / AuNCs-iRGD nanoparticles.

[0032] Example 4: An electrochemical detection sensor was constructed using the SiO2 / AuNCs-iRGD nanoparticles prepared in Example 1 to detect the concentration of doxorubicin in living cells. The method is as follows: (1) Modification of gold electrode: The purified gold electrode was immersed in a solution of HAuCl4 (final concentration 1.0 mg / mL) containing KNO3 (final concentration 0.1 M) and electrodeposited at -0.2 V for 20 seconds to obtain a gold nanoparticle modified electrode (AuNPs / Au). Then, the AuNPs / Au electrode was incubated in a 1 μM SH-DNA (TGG ATG ATG TGG TAT GGA AC-(CH2)6-SH) solution at room temperature for 16 hours to fix the DNA on the surface of the gold electrode through the Au-S bond between the SH-DNA and AuNPs, thus obtaining a DNA modified AuNPs / Au electrode.

[0033] The surface morphology and electrochemical detection performance of modified or unmodified gold electrodes were examined, and the results are as follows: Figure 3 As shown, Figure 3 Image A in the middle is a scanning electron microscope image of a bare gold electrode (Au electrode); Figure 3 Figure B in the middle is a scanning electron microscope image of the AuNPs-modified electrode (AuNPs / Au electrode); Figure 3 Figure C shows the AC impedance diagrams of the Au electrode and the AuNPs / Au electrode; Figure 3 Figure D shows the cyclic voltammograms of the Au electrode and the AuNPs / Au electrode.

[0034] Figure 3 The middle C figure shows that the reduction peak potential of doxorubicin cyclic voltammetry appears at -0.56V. After electrodeposition of AuNPs, compared with the bare gold electrode, the peak current signal is significantly increased and the impedance is decreased. Figure 3 As shown in Figure D, this is because AuNPs significantly increase the specific surface area of ​​the gold electrode, enabling it to be modified with more DNA, thereby binding more doxorubicin molecules.

[0035] (2) Assembly of breast cancer cells on the electrode surface Human breast cancer cells MCF-7 were cultured in complete DMEM medium at 37°C in a 5% CO2 incubator. After cell expansion, the cells were digested with trypsin containing EDTA for 5 min, centrifuged at 1000 rpm for 5 min, and the cell concentration was adjusted to 1×10⁶ cells / mL with DMEM medium. 6Cells were collected and prepared. Then, 100 μL of 1×PBS solution containing SiO2 / AuNCs-iRGD (containing a final concentration of 50 μM iRGD peptide) and 100 μL of 1×PBS solution containing a final concentration of 50 μM doxorubicin were added to 2000 μL of the above cell suspension, and incubated at 37°C for 2 hours. The supernatant was discarded by centrifugation, and the precipitate was washed with PBS and transferred to the appropriate test tubes. 900 μL of culture medium was added, and after centrifugation for 5 min, the supernatant was removed. 300 μL of culture medium was added to each tube to resuspend the cells (in another parallel experiment, 300 μL of trypsin was added to each well to lyse the cells), obtaining a cell suspension sample. Finally, the DNA-modified AuNPs / Au electrode was inserted into the cell suspension sample and incubated at 37°C for 6 h, dynamically monitoring impedance changes. The assembly of cells on the electrode surface was characterized using electrochemical impedance spectroscopy, and the results are as follows: Figure 4 As shown. After DNA modification, the impedance of the AuNPs / Au electrode increases because the groups on the DNA surface hinder electron transfer (e.g., Figure 4 (As shown in curve a). When SiO2 / AuNCs-iRGD is added, the cells are immobilized on the AuNPs / Au electrode surface by DNA strands, which to some extent shields the DNA groups, thus reducing the impedance (e.g., ...). Figure 4 (As shown in curve b). After trypsin digestion, the cells on the AuNPs / Au electrode surface are lysed, DNA groups are re-exposed, and the impedance begins to increase again, but it cannot return to the initial state (e.g., ...). Figure 4 (As shown in curve c), because doxorubicin interacts with DNA, neutralizing some of the charge.

[0036] (3) Investigation of methods for intracellular detection of doxorubicin concentration The concentration of the MCF-7 cell suspension was adjusted to 1×10⁻⁶. 6 / mL, 9 replicates, each with 2000 μL LCF-7 cell suspension. Three replicates were each given 100 μL of 1×PBS solution of iRGD (final iRGD peptide concentration 50 μM), three replicates were each given 100 μL of 1×PBS solution of SiO2 / AuNCs (final concentration 1 mg / mL), and the other three replicates were each given 100 μL of 1×PBS solution of SiO2 / AuNCs-iRGD (final concentration 1 mg / mL). Nine cell suspensions were then added to 100 μL of 1×PBS solution containing 50 μM doxorubicin, incubated at 37°C for 2 hours, centrifuged, and the precipitate was collected. The cells were washed three times with PBS, and 900 μL of culture medium was added. After centrifugation for 5 minutes, the supernatant was removed, and each tube was resuspended in 300 μL of culture medium. A portion of the cell suspension samples was used for laser confocal imaging, and DNA-modified AuNPs / Au electrodes were inserted into the remaining cell suspension samples. The samples were incubated at 37°C for 6 hours. The incubated DNA-modified AuNPs / Au electrodes were then removed and transferred to Tris-HCl buffer (pH 6.8) for electrochemical detection. A three-electrode system was used: a saturated calomel electrode as the reference electrode; a modified gold electrode as the working electrode; and a platinum wire electrode as the auxiliary electrode. Shanghai Chenhua Electrochemical Workstation CHI660E was used. DPV parameters: Init E:-0.3, Final E:-1, Incr E:0.004, Amplitude:0.05, Pulse Width:0.05, Sampling Width:0.0167, Pulse Period:0.5, Quiet Time:2, Sensitivity:1·e -004 ).

[0037] Laser confocal imaging results as follows Figure 5 As shown, the sample with only iRGD peptide added showed almost no red fluorescence, but the detected electrochemical signal peak of doxorubicin was strong. After removing the background signal, the average difference in current intensity (I-I0) was -33.06 μA. This is because iRGD peptide has membrane permeability and active targeting, which can bind to the cell membrane and promote the entry of doxorubicin into the cell, but it does not emit red fluorescence. The sample with added SiO2 / AuNCs showed a small amount of red fluorescence, but the I-I0 value was very small, with an average value of -0.18 μA. The composite nanoparticles SiO2 / AuNCs have passive targeting and a certain affinity for cells, but without iRGD peptide modification, their ability to penetrate cell membranes is poor. Therefore, a small amount of red light can be observed around the cells. However, doxorubicin in the cell suspension can hardly enter the cells, and after washing with PBS, the drug is almost completely removed. The sample with added SiO2 / AuNCs-iRGD showed strong red fluorescence and a large I-I0 value, with an average value of -32.02 μA. SiO2 / AuNCs-iRGD possesses good targeting and membrane permeability, facilitating the entry of extracellular drugs into cells while also providing fluorescence imaging capabilities, thus integrating treatment, diagnosis, and detection.

[0038] (4) Optimization of SiO2 / AuNCs-iRGD addition concentration To investigate the effect of SiO2 / AuNCs-iRGD on the electrochemical detection of doxorubicin in cells, 100 μL of 1×PBS solution of SiO2 / AuNCs-iRGD (equivalent to final concentrations of 10, 25, 50, 80, 100, and 200 μM iRGD peptide) and 100 μL of 1×PBS solution of doxorubicin at a final concentration of 50 μM were added to six identical MCF-7 cell suspensions (1×10⁻⁶ cells / mL). 6 Cells / mL (2000 μL per group) were incubated at 37°C for 2 hours. The precipitate was then centrifuged, washed three times with PBS, and 900 μL of culture medium was added. After centrifugation for 5 minutes, the supernatant was removed, and 300 μL of culture medium was added to each tube to resuspend the cells and obtain a cell suspension sample. A portion of the cell suspension sample was used for cell viability and uptake assays. Simultaneously, a DNA-modified AuNPs / Au electrode was inserted into the remaining cell suspension sample and incubated for another 6 hours. The incubated electrode was then removed and transferred to Tris-HCl buffer (pH 6.8) for DPV measurement (parameters: Init E: -0.3, Final E: -1, Incr E: 0.004, Amplitude: 0.05, Pulse Width: 0.05, Sampling Width: 0.0167, Pulse Period: 0.5, Quiet Time: 2, Sensitivity: 1·e). -004 ).

[0039] The results are as follows Figure 6 As shown, Figure 6 Figure A shows the effect of SiO2 / AuNCs-iRGD concentration on the survival rate of breast cancer MCF-7 cells; Figure 6 Figure B shows the effect of SiO2 / AuNCs-iRGD addition concentration on the uptake of SiO2 / AuNCs-iRGD nanoparticles by MCF-7 cells; Figure 6 Figure C shows the effect of SiO2 / AuNCs-iRGD addition concentration on DPV signal intensity.

[0040] Figure 6Figure A shows that, compared to free iRGD, SiO2 / AuNCs-iRGD containing 10-100 μM iRGD had less impact on breast cancer cell survival, while the composite nanoparticle suspension containing 200 μM iRGD caused some damage to the cells, leading to cell death. Furthermore, combined with laser confocal imaging (… Figure 6 Figure B in the middle) and the results of DPV measurement ( Figure 6 (See Figure C). Finally, SiO2 / AuNCs-iRGD containing a final concentration of 100 μM iRGD peptide was selected as the subsequent detection condition.

[0041] (5) Quantitative detection of doxorubicin concentration in breast cancer cells using electrochemical methods Following the above method, 100 μL of 1×PBS solution containing iRGD-SiO2 / AuNCs (containing a final concentration of 100 μM iRGD peptide) and 100 μL of 1×PBS solution containing different concentrations of doxorubicin (final concentrations of 0.05, 0.1, 0.5, 1, 5, 10, 25, 50, 100, and 200 μM) were mixed with MCF-7 cell suspension (1×10⁻⁶). 6 Cells / mL (2000 μL per group) were co-cultured at 37℃ for 2 h. After centrifugation, the precipitate was collected, washed three times with PBS, and 900 μL of culture medium was added. After centrifugation for 5 min, the supernatant was removed, and 300 μL of culture medium was added to each tube to resuspend the cell suspension. The DNA-modified AuNPs / Au electrode was inserted into the cell suspension samples of each group and incubated for another 6 h. Then, the incubated electrode was removed and transferred to Tris-HCl buffer (pH 6.8) for DPV measurement to investigate the relationship between the current intensity of the DPV signal and the concentration of doxorubicin.

[0042] The results are as follows Figure 7 As shown, Figure 7 Figure A shows the DPV response within the range of 0.05-200 μM after adding doxorubicin; Figure 7 Figure B in the middle plot is a linear fit plot of the DPV signal intensity and the logarithmic value of doxorubicin concentration.

[0043] Figure 7 Figure A shows that with increasing doxorubicin concentration, more drug can enter cells mediated by iRGD-SiO2 / AuNCs, and live cells are immobilized on the electrode by iRGD-SiO2 / AuNCs. The electrochemical signal of doxorubicin transduced to the extracellular space via iRGD-SiO2 / AuNCs and DNA also increases accordingly. Within the range of 0.05–200 μM, the current intensity of DPV shows a good linear relationship with the logarithm of doxorubicin concentration (e.g., ...). Figure 7 (As shown in Figure B). The linear regression equation is y = -2.9187 - 27.1248x, R02 =0.9981, detection limit is 0.01μM. Where the y-axis is the current intensity value I of the DPV signal of doxorubicin, and the x-axis is the logarithm of the concentration of doxorubicin lgC.

[0044] It should be noted that, in the investigation of intracellular doxorubicin detection methods, after the applicant increased the final concentration of the initial doxorubicin addition to 500 μM, it was found that within a range not exceeding 400 μM, almost all of the initially added doxorubicin entered the cells, and no obvious doxorubicin signal could be detected in the extracellular wash solution. Therefore, within the range of 0.05-200 μM, the added doxorubicin can completely enter the cells. Accordingly, in this invention, the initial concentration of added doxorubicin is used to replace the actual intracellular doxorubicin concentration to establish a linear relationship with the DPV electrical signal.

Claims

1. A nanoparticle, characterized in that, The nano-carrier particle comprises at least one of SiO2 nanoparticles and gold nanoparticles, and the particle size of the nano-carrier particle is not more than 300 nm; the gold nanocluster is coupled to the surface of the nano-carrier particle, and the gold nanocluster and / or the nano-carrier particle is coupled to the surface of the amino acid sequence iRGD peptide shown in SEQ ID No.

1.

2. The nanoparticle of claim 1, wherein, In step (1), the nano-carrier particle comprises at least one of SiO2 nanoparticles and gold nanoparticles, and the particle size of the nano-carrier particle is not more than 300 nm; the particle size of the gold nanocluster is not more than 15 nm.

3. The method for preparing nanoparticles according to claim 1 or 2, characterized in that, The method comprises the following steps: The nano-carrier particle coupled to the gold nanocluster is dispersed in a buffer solution containing a coupling agent, and then the iRGD peptide is added after activation; after stirring and reaction, the precipitate is filtered and washed to obtain the composite nanoparticle modified with the iRGD.

4. The method for preparing nanoparticles according to claim 3, characterized in that, The preparation method of the nano-carrier particle coupled to the gold nanocluster comprises the following steps: The amino-modified SiO2 nanoparticles are dispersed into the gold nanocluster, and then N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide are added for activation and continuous stirring and reaction; after centrifugation and washing, the silica / gold nanocluster composite fluorescent nanoparticle is obtained.

5. The method for preparing nanoparticles according to claim 3, characterized in that, The coupling agent is N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and the mass ratio of the nano-carrier particle coupled to the gold nanocluster to the iRGD peptide is 3-7:1; the stirring and reaction condition is 100-200 rpm, stirring and reaction at room temperature for 12-48 h in the dark.

6. The application of the nanoparticle of claim 1 or 2 in electrochemical detection of intracellular doxorubicin concentration or fluorescence imaging of living cells.

7. Use according to claim 6, characterized in that, The method comprises the following steps: (1) The nanoparticle and doxorubicin with different final concentrations are added to a living cell suspension, and then the unbound nanoparticle and doxorubicin are removed by centrifugation and washing after incubation to obtain the cells to be detected; (2) The cells to be detected are resuspended in a culture medium to obtain a cell suspension to be detected, and then the cell suspension to be detected is subjected to laser confocal imaging; or a gold electrode modified with the DNA sequence shown in SEQ ID No. 2 is inserted into the cell suspension to be detected, and then the gold electrode is transferred to a buffer solution for electrochemical detection after incubation; a linear relationship between the measured electrochemical signal and the intracellular doxorubicin concentration of the cells treated with different final concentrations of doxorubicin is established, and the intracellular doxorubicin concentration is calculated according to the linear relationship and the electrochemical signal of the sample of the cells to be detected.

8. Use according to claim 7, characterized in that, In step (1), the final concentration of doxorubicin ranges from 0.05 to 200 μM, and the molar ratio of the iRGD peptide on the surface of the nanoparticle to doxorubicin is 1-20:5; the incubation condition is 36-38℃ for 1-4 h; In step (2), the electrochemical signal is the current intensity measured by differential pulse voltammetry, and the incubation condition is 36-38℃ for 3-9 h; the buffer solution is Tris-HCl buffer solution; The cells are tumor cells.

9. Use according to claim 7, characterized in that, The preparation method of the gold electrode modified with the DNA sequence comprises the following steps: The gold electrode is immersed in a HAuCl4 solution containing KNO3, and then electrodeposition is performed at -0.1~-0.3 V for 10-30 s to obtain an AuNPs / Au electrode. The AuNPs / Au electrode is incubated in a 0.5-1.5 μM SH-DNA solution for 8-24 h to obtain a gold electrode surface-modified with a DNA sequence.

10. An electrochemical sensor for detecting intracellular doxorubicin concentration, comprising: The gold electrode surface-modified with the DNA sequence shown in SEQ ID No. 2, a reference electrode, a counter electrode, an incubation tank containing an incubation solution containing the nanoparticles of claim 1 or 2, and an electrochemical detection tank containing a buffer solution.

Images