Chiral peg-ag6d / lsp6 cluster-based nanoparticles, methods and uses

Abstract

The application discloses a kind of chiral PEG-Ag6D / LSP6 cluster-based nanoparticles, method and application, method includes: S1, the preparation of chiral nanocluster, in mixed solvent silver salt and thiazolidine thione ligand is added, and the crystal of chiral silver nanocluster is obtained by volatilization;S2, a certain amount of amphiphilic polymer DSPE-PEG2000 and chiral silver nanocluster are dissolved in organic solvent, then added to a certain volume of aqueous solution;S3, under the condition of violent stirring, reaction is carried out until organic solvent is completely removed, filtration is carried out, and the product is obtained;The chiral PEG-Ag6D / LSP6 cluster-based nanoparticles prepared in the application has good biocompatibility, rapid response to biological thiol, strong anti-interference ability and low detection limit, and is suitable for high-throughput equipment, can be used for biological thiol distinction, enantioselective chiral recognition and biological thiol quantitative detection in plasma, and as potential nanomedicine and exhibit chiral-dependent anti-tumor effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a chiral PEG-Ag6D / LSP6 cluster-based nanoparticle, its method, and its application. Background Technology

[0002] Biothiols, such as cysteine ​​(Cys), homocysteine ​​(Hcy), and glutathione (GSH), are associated with cancer, autoimmune diseases, cardiovascular diseases, and neurodegenerative diseases. Quantifying the concentration of biothiols in bodily fluids is crucial for the early diagnosis of these diseases and for personalized treatment of patients. However, selective biothiol analysis is particularly challenging due to the similarity of their chemical structures and properties. Cys, Hcy, and GSH, along with other amino acids, often interfere with each other in molecular recognition processes, further complicating substrate-specific detection and enantioselective quantification. Although various quantitative methods have been applied to biothiol monitoring, including fluorescence, colorimetric / UV, and electrochemical methods, most can only identify one or two biothiols, and sensors that can effectively distinguish all three remain lacking. To date, fluorescence-based small molecule compounds have been developed to distinguish the three biothiols, but these require complex molecular designs and a strong chemical foundation. Furthermore, their preparation conditions are often harsh and cumbersome, resulting in high costs, high toxicity, and poor stability. Therefore, it is necessary to develop novel strategies with high sensitivity, good biocompatibility, and simple synthetic methods for the detection of biothiols in complex physiological environments. Chirality plays a crucial role in biology, and the presence of D-amino acids in organisms is a useful indicator of various processes, including aging, disease, or disorders. However, many amino acids have small molecular weights and high polarity, and do not contain large aromatic groups. Determining their configuration and ee value using optical methods is extremely challenging. Therefore, it is highly desirable to develop a biosensor with good biocompatibility, rapid response to biothiols, strong anti-interference ability, low detection limit, and suitability for high-throughput devices, which can be used for the differentiation of biothiols, enantioselective chiral recognition, and quantitative detection of biothiols in plasma.

[0003] The biological functions of supramolecular assemblies such as lipids, proteins, and DNA are highly dependent on their chirality. Unlike molecular chirality, the biological activities of supramolecular chirality have not been fully studied. In recent years, the biological applications of artificial supramolecular chiral materials have received widespread attention. Differences in the chirality of supramolecular nanomaterials have significant effects on protein adsorption, cell adhesion, cell proliferation and differentiation, phagocytosis, apoptosis, antigen processing and presentation, and disease diagnosis and treatment. This supports the importance of chirality at the interface between biomaterials and organisms. However, the mechanistic explanation for the chirality-dependent effects of biological systems remains unclear. Nanoclusters, with their atomically precise structures and specific interaction sites, can further contribute to our understanding of chirality-dependent effects. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a chiral PEG-Ag6D / LSP6 cluster-based nanoparticle and a method. The chiral PEG-Ag6D / LSP6 cluster-based nanoparticle can be used as a nanosensor for the differentiation of biothiols, enantioselective chiral recognition, and quantitative detection of biothiols in plasma. Furthermore, the chiral PEG-Ag6D / LSP6 cluster-based nanoparticle can also serve as a potential nanomedicine for chiral-dependent antitumor drugs.

[0005] To achieve this objective, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing chiral PEG-Ag6D / LSP6 cluster-based nanoparticles, comprising the following steps:

[0007] S1. Preparation of chiral nanoclusters: Silver salt and ligands of thiazolidinthone were added to a mixed solvent, and the mixture was volatilized to obtain crystals of chiral silver nanoclusters.

[0008] S2. A certain amount of the amphiphilic polymer DSPE-PEG2000 and the chiral silver nanoclusters obtained in step S1 are dissolved in an organic solvent and then added to a certain volume of aqueous solution to prepare water-soluble chiral cluster-based nanoparticles through self-assembly.

[0009] S3. The reaction was carried out under vigorous stirring until the organic solvent was completely removed. The reaction was then stopped, and the product was filtered to obtain biocompatible chiral PEG-Ag6D / LSP6 cluster-based nanoparticles.

[0010] Preferably, in step S1, the silver salt is selected from one or more combinations of AgNO3, AgBF4, AgF6P and CF3SO3Ag, and the mixed solvent is selected from one or more combinations of DMF, DMA, DMSO, CH3CN, CH2Cl2, n-hexane and THF.

[0011] Preferably, in step S2, the initial concentration of the chiral silver nanoclusters is 0.1-0.5 mg / mL; more preferably, in step 2, the initial concentration of the chiral silver nanoclusters is 0.1 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL.

[0012] Preferably, the mass ratio of silver nanoclusters to amphiphilic polymer DSPE-PEG2000 is 1:(1-10); more preferably, the mass ratio of silver nanoclusters to amphiphilic polymer DSPE-PEG2000 is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8 or 1:10.

[0013] Preferably, in step S2, the organic solvent includes THF and CH2Cl2, the aqueous solution is ultrapure water, and the volume ratio of organic solvent to ultrapure water is 1:(5-20); more preferably, the volume ratio of organic solvent to ultrapure water is 1:5, 1:8, 1:10, 1:15 or 1:20.

[0014] Preferably, in step S3, the stirring speed is 400 r / min–1200 r / min, the time for complete removal of organic solvent is between 18 h and 36 h, and a 0.22 μm needle filter is used for filtration.

[0015] In a second aspect, the present invention provides a chiral PEG-Ag6D / LSP6 cluster-based nanoparticle, wherein the morphology of the chiral PEG-Ag6D / LSP6 cluster-based nanoparticle is spherical or ellipsoidal, and the particle size is 80 nm–110 nm, as shown in a transmission electron microscope image.

[0016] Preferably, the chiral PEG-Ag6D / LSP6 cluster-based nanoparticles have a spherical or ellipsoidal morphology, an average particle size of 81±0.1 nm, good dispersibility in aqueous solution, a hydrated particle size of 82.5 nm, and a dispersion coefficient PDI=0.11.

[0017] Preferably, the chiral PEG-Ag6D / LSP6 cluster-based nanoparticles are prepared using the method described in this invention.

[0018] Preferred method includes the following steps:

[0019] S1. Preparation of chiral nanoclusters: Silver salt and ligands of thiazolidinthone were added to a mixed solvent, and the mixture was volatilized to obtain crystals of chiral silver nanoclusters.

[0020] S2. A certain amount of the amphiphilic polymer DSPE-PEG2000 and the chiral silver nanoclusters obtained in step S1 are dissolved in an organic solvent and then added to a certain volume of aqueous solution to prepare water-soluble chiral cluster-based nanoparticles through self-assembly.

[0021] S3. The reaction was carried out under vigorous stirring until the organic solvent was completely removed. The reaction was then stopped, and the product was filtered to obtain biocompatible chiral PEG-Ag6D / LSP6 cluster-based nanoparticles.

[0022] Preferably, in step S1, the silver salt is selected from one or more combinations of AgNO3, AgBF4, AgF6P and CF3SO3Ag, and the mixed solvent is selected from one or more combinations of DMF, DMA, DMSO, CH3CN, CH2Cl2, n-hexane and THF.

[0023] Preferably, in step S2, the initial concentration of the chiral silver nanoclusters is 0.1-0.5 mg / mL; more preferably, in step 2, the initial concentration of the chiral silver nanoclusters is 0.1 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL.

[0024] Preferably, the mass ratio of silver nanoclusters to amphiphilic polymer DSPE-PEG2000 is 1:(1-10); more preferably, the mass ratio of silver nanoclusters to amphiphilic polymer DSPE-PEG2000 is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8 or 1:10.

[0025] Preferably, in step S2, the organic solvent includes THF and CH2Cl2, the aqueous solution is ultrapure water, and the volume ratio of organic solvent to ultrapure water is 1:(5-20); more preferably, the volume ratio of organic solvent to ultrapure water is 1:5, 1:8, 1:10, 1:15 or 1:20.

[0026] Preferably, in step S3, the stirring speed is 400 r / min–1200 r / min, the time for complete removal of organic solvent is between 18 h and 36 h, and a 0.22 μm needle filter is used for filtration.

[0027] A third aspect of the present invention provides the application of chiral PEG-Ag6D / LSP6 cluster-based nanoparticles prepared by the method of the present invention, or the chiral PEG-Ag6D / LSP6 cluster-based nanoparticles of the present invention, in the preparation of reagents for distinguishing biothiols, enantioselective chiral recognition, and quantitative detection of biothiols in plasma.

[0028] Preferably, the application includes the following steps:

[0029] R1. Dissolve chiral PEG-Ag6D / LSP6 cluster-based nanoparticles in solvent A to prepare a stock solution of a specific concentration, and then dilute it to an appropriate concentration for later use.

[0030] R2. Take the sample to be tested, add the diluent prepared in step R1, and then determine the type and content of biothiols in the sample by observing the presence and changes of the CD signal.

[0031] Preferably, solvent A includes ultrapure water or phosphate buffer.

[0032] A chiral PEG-Ag6D / LSP6 cluster-based nanoparticle, as shown in a transmission electron microscope image, has a spherical or ellipsoidal morphology and a particle size of 80 nm–110 nm.

[0033] Preferably, the chiral PEG-Ag6D / LSP6 cluster-based nanoparticles have a spherical or ellipsoidal morphology, an average particle size of 81±0.1 nm, good dispersibility in aqueous solution, a hydrated particle size of 82.5 nm, and a dispersion coefficient PDI=0.11.

[0034] Preferably, the chiral PEG-Ag6D / LSP6 cluster-based nanoparticles are prepared using the method described in this invention.

[0035] Preferred method includes the following steps:

[0036] S1. Preparation of chiral nanoclusters: Silver salt and ligands of thiazolidinthone were added to a mixed solvent, and the mixture was volatilized to obtain crystals of chiral silver nanoclusters.

[0037] S2. A certain amount of the amphiphilic polymer DSPE-PEG2000 and the chiral silver nanoclusters obtained in step S1 are dissolved in an organic solvent and then added to a certain volume of aqueous solution to prepare water-soluble chiral cluster-based nanoparticles through self-assembly.

[0038] S3. The reaction was carried out under vigorous stirring until the organic solvent was completely removed. The reaction was then stopped, and the product was filtered to obtain biocompatible chiral PEG-Ag6D / LSP6 cluster-based nanoparticles.

[0039] Preferably, in step S1, the silver salt is selected from one or more combinations of AgNO3, AgBF4, AgF6P and CF3SO3Ag, and the mixed solvent is selected from one or more combinations of DMF, DMA, DMSO, CH3CN, CH2Cl2, n-hexane and THF.

[0040] Preferably, in step S2, the initial concentration of the chiral silver nanoclusters is 0.1-0.5 mg / mL; more preferably, in step 2, the initial concentration of the chiral silver nanoclusters is 0.1 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL.

[0041] Preferably, the mass ratio of silver nanoclusters to amphiphilic polymer DSPE-PEG2000 is 1:(1-10); more preferably, the mass ratio of silver nanoclusters to amphiphilic polymer DSPE-PEG2000 is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8 or 1:10.

[0042] Preferably, in step S2, the organic solvent includes THF and CH2Cl2, the aqueous solution is ultrapure water, and the volume ratio of organic solvent to ultrapure water is 1:(5-20); more preferably, the volume ratio of organic solvent to ultrapure water is 1:5, 1:8, 1:10, 1:15 or 1:20.

[0043] Preferably, in step S3, the stirring speed is 400 r / min–1200 r / min, the time for complete removal of organic solvent is between 18 h and 36 h, and a 0.22 μm needle filter is used for filtration.

[0044] A fourth aspect of the present invention provides the application of chiral PEG-Ag6D / LSP6 cluster-based nanoparticles prepared by the method of the present invention, or the chiral PEG-Ag6D / LSP6 cluster-based nanoparticles of the present invention, in the preparation of antitumor drugs.

[0045] A chiral PEG-Ag6D / LSP6 cluster-based nanoparticle, as shown in a transmission electron microscope image, has a spherical or ellipsoidal morphology and a particle size of 80 nm–110 nm.

[0046] Preferably, the chiral PEG-Ag6D / LSP6 cluster-based nanoparticles have a spherical or ellipsoidal morphology, an average particle size of 81±0.1 nm, good dispersibility in aqueous solution, a hydrated particle size of 82.5 nm, and a dispersion coefficient PDI=0.11.

[0047] Preferably, the chiral PEG-Ag6D / LSP6 cluster-based nanoparticles are prepared using the method described in this invention.

[0048] Preferred method includes the following steps:

[0049] S1. Preparation of chiral nanoclusters: Silver salt and ligands of thiazolidinthone were added to a mixed solvent, and the mixture was volatilized to obtain crystals of chiral silver nanoclusters.

[0050] S2. A certain amount of the amphiphilic polymer DSPE-PEG2000 and the chiral silver nanoclusters obtained in step S1 are dissolved in an organic solvent and then added to a certain volume of aqueous solution to prepare water-soluble chiral cluster-based nanoparticles through self-assembly.

[0051] S3. The reaction was carried out under vigorous stirring until the organic solvent was completely removed. The reaction was then stopped, and the product was filtered to obtain biocompatible chiral PEG-Ag6D / LSP6 cluster-based nanoparticles.

[0052] Preferably, in step S1, the silver salt is selected from one or more combinations of AgNO3, AgBF4, AgF6P and CF3SO3Ag, and the mixed solvent is selected from one or more combinations of DMF, DMA, DMSO, CH3CN, CH2Cl2, n-hexane and THF.

[0053] Preferably, in step S2, the initial concentration of the chiral silver nanoclusters is 0.1-0.5 mg / mL; more preferably, in step 2, the initial concentration of the chiral silver nanoclusters is 0.1 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL.

[0054] Preferably, the mass ratio of silver nanoclusters to amphiphilic polymer DSPE-PEG2000 is 1:(1-10); more preferably, the mass ratio of silver nanoclusters to amphiphilic polymer DSPE-PEG2000 is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8 or 1:10.

[0055] Preferably, in step S2, the organic solvent includes THF and CH2Cl2, the aqueous solution is ultrapure water, and the volume ratio of organic solvent to ultrapure water is 1:(5-20); more preferably, the volume ratio of organic solvent to ultrapure water is 1:5, 1:8, 1:10, 1:15 or 1:20.

[0056] Preferably, in step S3, the stirring speed is 400 r / min–1200 r / min, the time for complete removal of organic solvent is between 18 h and 36 h, and a 0.22 μm needle filter is used for filtration.

[0057] In summary, this invention provides a chiral PEG-Ag6D / LSP6 cluster-based nanoparticle as a nanosensor for the differentiation of biothiols, enantioselective chiral recognition, quantitative detection of biothiols in plasma, and as a potential nanomedicine for chiral-dependent antitumor applications. The chiral PEG-Ag6D / LSP6 cluster-based nanoparticles are characterized by: chiral silver nanoclusters formed from silver salt and thiazolidinthione (D / LSP) ligands, followed by the self-assembly of the silver nanoclusters with the amphiphilic polymer DSPE-PEG2000 to construct a biocompatible chiral nanomaterial. The Ag6DSP6 nanoclusters have a spherical microstructure exhibiting distinct lattice fringes, and the average particle size of the Ag6DSP6 nanoclusters is 2.1 ± 0.02 nm. PEG-Ag6D / LSP6 exhibits a spherical or ellipsoidal morphology with a particle size of 80 nm–110 nm (average particle size 81 ± 0.1 nm). It demonstrates good dispersibility in aqueous solution, with an average hydrated particle size of approximately 82.5 nm and an average dispersion index (PDI) of approximately 0.11. Adding different biothiols to the PEG-Ag6DSP6 solution produces different CD response modes, allowing for enantioselective chiral recognition of biothiols by observing changes in their CD signals. Furthermore, PCA analysis can be used to differentiate the three biothiols and quantify their presence in plasma. Cytotoxicity experiments demonstrated that D- or L-configured nanoparticles did not inhibit cell proliferation or produce cytotoxicity in normal cells at the same concentration. However, they exhibited significant inhibitory effects on cancer cells, with the D-configuration showing superior efficacy compared to the L-configuration.

[0058] Compared with the prior art, the beneficial effects and significant progress of applying the technical solution of the present invention are as follows:

[0059] 1. Compared with traditional nanomaterials, the chiral PEG-Ag6D / LSP6 cluster-based nanoparticles prepared in this invention have atomically precise structures, good biocompatibility, fast response speed and strong anti-interference ability, making them suitable for the detection of biothiols in complex blood environments and meeting the needs of practical applications.

[0060] 2. The chiral PEG-Ag6D / LSP6 cluster-based nanoparticles provided by this invention are easy to prepare and suitable for high-throughput equipment, unlike the complex operation of existing commercial kits for the detection of biothiols.

[0061] 3. The chiral PEG-Ag6D / LSP6 cluster-based nanoparticles provided in this invention can be used simultaneously for the differentiation of biothiols, enantioselective chiral recognition, and quantitative detection of biothiols in plasma. This is the first time that a nanosensor with these three properties has been realized.

[0062] 4. The chiral PEG-Ag6D / LSP6 cluster-based nanoparticles prepared by this invention have chirality-dependent antitumor effects. The precise structure and specific active sites of the nanoclusters provide support for exploring the interaction between chirality and the interface between biomaterials and organisms. Attached Figure Description

[0063] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the embodiments of the present invention will be briefly described below.

[0064] Figure 1 These are transmission electron microscope (TEM) images of the chiral nanoclusters and PEG-modified cluster-based nanoparticles prepared in this invention.

[0065] Figure 2 The images show dynamic light scattering (DLS) patterns of the PEG-Ag6DSP6 nanomaterials prepared according to this invention; where a) is the dynamic light scattering (DLS) pattern of PEG-Ag6DSP6, b) is the particle size distribution of PEG-Ag6DSP6 prepared at different initial concentrations, and c) is the particle size distribution of PEG-Ag6DSP6 prepared at different feed mass ratios.

[0066] Figure 3 This is a chiral recognition diagram of the corresponding selectivity of the PEG-Ag6DSP6 nanomaterial prepared in this invention to biothiols; wherein, a) the change of CD spectrum of PEG-Ag6DSP6 detecting the same concentration of L-Cys, b) the change of CD spectrum of PEG-Ag6DSP6 detecting the same concentration of D-Cys, and c) the relationship between Cys concentration and the change of CD signal at 275nm.

[0067] Figure 4 The diagram shows the CD mode response of the PEG-Ag6DSP6 nanomaterial prepared in this invention to three biothiols and the differentiation of the three biothiols by PCA analysis; wherein, a) CD spectral change diagram of PEG-Ag6DSP6 detecting L-Cys, b) CD spectral change diagram of PEG-Ag6DSP6 detecting L-GSH, c) CD spectral change diagram of PEG-Ag6DSP6 detecting L-Hcy, and d) PCA score diagram according to different change modes.

[0068] Figure 5 This is a test image of the anti-interference ability of the PEG-Ag6DSP6 nanomaterial prepared in this invention;

[0069] Figure 6The graph shows the detection of biothiols in plasma using the PEG-Ag6DSP6 nanomaterial prepared in this invention; where a) is the blood pretreatment process, b) is the result of the commercial standard method for detecting GSH concentration in plasma, and c) is the result of PEG-Ag6DSP6 detecting GSH concentration in plasma.

[0070] Figure 7 This invention presents a cytotoxicity experiment of a pair of enantiomers of nanoclusters against cancer cells and normal cells; wherein, a) the relative cell activity of PEG-Ag6D / LSP6 nanoparticles after incubation with A549 cells, and b) the relative cell activity of PEG-Ag6D / LSP6 nanoparticles after incubation with BEAS-2B cells. Detailed Implementation

[0071] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this invention, those skilled in the art can make various alterations and modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0072] Furthermore, the reaction apparatus and chemical reagents involved in the following examples and comparative examples are all commercially available, as are the detection instruments and detection reagents involved.

[0073] The following embodiments and comparative examples were tested using the following methods, equipment, or standards:

[0074] Transmission electron microscopy (TEM) testing: The prepared silver nanoclusters were ground into powder, then ultrasonically dispersed in ethanol for 10 min, and 5 μL was suspended and dropped onto a copper grid. The prepared chiral cluster-based nanoparticles were dissolved in ultrapure water to prepare a 0.01 M stock solution, which was then diluted 100 times, and 5 μL was suspended and dropped onto a copper grid. The mixture was dried in a vacuum drying oven at 60 °C and observed under a TEM.

[0075] Hydrated particle size test: First, prepare the sample to be tested into a 0.1 mM aqueous solution, then take 30 μL and dilute it to 3 mL and place it in a dynamic light scattering instrument to measure its hydrated particle size in the aqueous solution;

[0076] Circular dichroism (CD) spectroscopy: The prepared chiral cluster-based nanoparticles were dissolved in phosphate-buffered saline (1x PBS, pH=7.4) to prepare a stock solution with a concentration between 1M and 0.1M. This stock solution was then diluted 10–100 times for use, with a volume of 3 mL. A stock solution of biothiols was prepared at 0.001 mM–2 mM. 3 μL of this stock solution was added to the 3 mL nanocluster solution. The CD spectrum was detected after 3–5 min.

[0077] Example 1

[0078] This embodiment provides a method for preparing nanoparticles based on chiral nanoclusters.

[0079] First, prepare the reactor equipment: Soak a 20mL flat-bottomed glass bottle in freshly prepared aqua regia for a period of time, then rinse it with distilled water, dry it in an oven, and then let it cool before use. The reactor equipment can be a glass bottle, a round-bottomed flask, or other reactor equipment.

[0080] The preparation of nanoparticles based on chiral nanoclusters in experimental groups 1-13 includes the following steps:

[0081] Step 1: The preparation of chiral nanoclusters is carried out using a simple one-step method. Silver nanoclusters are obtained by slowly evaporating silver salt and thiazolidinthione ligands in a mixed solvent in a dark environment at room temperature.

[0082] Step 2: Dissolve a certain amount of amphiphilic polymer DSPE-PEG2000 and chiral silver nanoclusters in a volatile organic solvent in different proportions, and then add them to a certain volume of ultrapure aqueous solution to prepare water-soluble chiral cluster-based nanoparticles through self-assembly.

[0083] Step 3: The reaction was carried out under vigorous stirring at room temperature until the organic solvent was completely removed. The reaction was then stopped, and the product was filtered through a filter membrane to obtain biocompatible chiral PEG-Ag6D / LSP6 cluster-based nanoparticles, which were then lyophilized for later use.

[0084] The preparation method employs the self-assembly of hydrophobic chiral silver nanoclusters and amphiphilic polymer DSPE-PEG2000 in aqueous solution to form biocompatible chiral cluster-based nanoparticles as biosensors.

[0085] As a preferred technical solution, further: the vigorous stirring speed in step 3 is 400 r / min–1200 r / min. The time for complete removal of the organic solvent is between 18 h and 36 h. The reaction solution is filtered using a 0.22 μm needle filter to remove large particles.

[0086] In experimental groups 1-13, the silver salt selected for preparing silver nanoclusters was AgBF4, the ligand was (S)-4-phenylthiazolidin-2-thione, and the mixed solvent was DMF and CH3CN. The initial concentration of silver nanoclusters varied among experimental groups when preparing PEG-modified biocompatible cluster nanoparticles (see Table 1 below). The mass ratio of silver nanoclusters to the amphiphilic polymer DSPE-PEG2000 also varied among experimental groups (see Table 1 below). Silver nanoclusters and DSPE-PEG2000 were dissolved in the volatile organic solvent THF, and the volume ratio of the organic solvent to ultrapure water was one of 1:5, 1:8, 1:10, 1:15, or 1:20.

[0087] Table 1

[0088]

[0089] Figure 1 and Figure 2 The images show transmission electron microscopy (TEM) and deep dispersion (DLS) images of the chiral PEG-Ag6DSP6 nanoparticles obtained in Experimental Group 1. TEM results indicate that the chiral PEG-Ag6DSP6 nanoparticles are spherical or ellipsoidal in shape, and are monodisperse nanoparticles with a diameter of approximately 80 nm. DLS results show that the hydrated particle size of the chiral nanoparticles ranges from 80 nm to 110 nm, with the smallest hydrated particle size (82.5 nm) only observed at an initial concentration of 0.25 mg / mL for the silver nanoclusters, which is consistent with the TEM results. Furthermore, DLS results also indicate that the chiral PEG-Ag6DSP6 nanoparticles exhibit good dispersibility in aqueous solution, with a dispersion index (PDI) of 0.11.

[0090] Example 2

[0091] This embodiment uses the chiral PEG-Ag6DSP6 nanoparticles synthesized in Experimental Group 1 of Example 1 to distinguish, enantioselectively recognize, and quantitatively detect biothiols in plasma. The experimental process includes the following steps:

[0092] Step 1: Dissolve the prepared chiral cluster-based nanoparticles in phosphate buffer (1x PBS, pH=7.4) to prepare a stock solution of a specific concentration. The concentration of the stock solution is between 1M and 0.1M. Dilute the stock solution 10 to 100 times for later use, with a volume of 3mL.

[0093] Step 2: Biothiols, mainly referring to cysteine ​​(Cys), glutathione (GSH), and homocysteine ​​(Hcy), are dissolved in PBS solution to prepare a 0.001mM–2mM stock solution. Then, 3μL is added to the above 3mL nanocluster solution. After 3–5 minutes, the CD spectrum is measured, and the CD signal values ​​at 275nm and 360nm are statistically analyzed.

[0094] Step 3: Blood is collected from healthy black mice and placed in a blood collection tube containing sodium EDTA. The mixture is stirred 3–5 times, allowed to stand for 10–15 minutes, and then centrifuged at 3000–12000 rpm for 5–15 minutes. The supernatant is collected for later use. Plasma is diluted 20–80 times for establishing calibration curves and subsequent measurements.

[0095] The experimental results are as follows:

[0096] See Figure 3When L-Cys and D-Cys of the same concentration (0–40 μM) were added to PEG-Ag6DSP6 solutions of fixed concentration, significant differences in CD signal changes were observed. When the concentration of L-Cys increased from 0 to 40 μM, the CD signal of the PEG-Ag6DSP6 solution at 275 nm decayed faster than that of the same amount of D-Cys. This indicates that the chiral nanocluster-based nanosensor enables enantioselective chiral recognition of biothiols.

[0097] See Figure 4 When three biothiols were added to a PEG-Ag6DSP6 solution of a fixed concentration, three modes of CD response appeared at 275 nm and 360 nm. By statistically analyzing the CD signal intensity at 275 nm and 360 nm and performing PCA analysis, the biothiols could be clearly distinguished.

[0098] See Figure 5 , Figure 6 As shown in Table 2, PEG-Ag6DSP6 exhibited good anti-interference ability and good detection capability of biothiol content in plasma samples from three groups of mice, comparable to commercially available reagents. Based on changes in the CD signal, PCA analysis can be used to determine the biothiol content in plasma samples, enabling timely treatment of diseases caused by abnormal biothiol levels. Compared to commercially available biothiol detection reagents, the chiral PEG-Ag6DSP6 nanoparticles of this invention are faster, more sensitive, more efficient, and easier to operate.

[0099] Table 2

[0100]

[0101] Example 3

[0102] This embodiment uses the chiral PEG-Ag6D / LSP6 nanoparticles synthesized in experimental group 1 of Example 1 as a potential nanomedicine for chiral-dependent antitumor effects.

[0103] Experimental methods

[0104] The prepared enantiomer nanoparticles were dispersed in phosphate-buffered saline (PBS) at a concentration of 50–100 μM and sonicated for 5 min for cytotoxicity assays. Specifically, cancer cells and normal cells were uniformly seeded in 96-well plates, with 100 μL of cell suspension per well, depending on the appropriate cell number (approximately 1–2 × 10⁴). Four to six replicates were performed for the same sample. The D or L-configuration nanoparticle solutions were co-incubated with the cells for 12–36 h. Then, 10 μL of CCK-8 was added to each well for further incubation for 0.5–4 h. The absorbance at 450 nm was measured using a microplate reader.

[0105] Experimental results:

[0106] See Figure 7 In cytotoxicity experiments on cancer cells and normal cells, nanoparticles of the same concentration had no effect on the growth of normal cells, but showed a significant inhibitory effect on the growth of cancer cells. Furthermore, the D-configuration nanoparticles exhibited a better inhibitory effect on cancer cells than the L-configuration, demonstrating a chirality-dependent antitumor effect.

[0107] In summary, this invention discloses the preparation of chiral nanoclusters and their application as nanosensors in the differentiation, enantioselective chiral recognition, and quantitative detection of biothiols in plasma, as well as their potential application as nanomedicines in chiral-dependent antitumor therapy. This relates to the fields of biosensor preparation, biosensor detection, and antitumor therapy. The preparation method utilizes silver nanoclusters and amphiphilic polymers through self-assembly, resulting in a simple, environmentally friendly, time-efficient, and high-yield process. The prepared chiral nanoparticles, with an average particle size of 82.5 nm, exhibit uniform size, good biocompatibility, rapid response to biothiols, strong anti-interference ability, and low detection limit, making them suitable for high-throughput equipment. They can be used for the differentiation, enantioselective chiral recognition, and quantitative detection of biothiols in plasma. In addition, the chiral nanocluster-based nanoparticles prepared in this invention also exhibit chirality-dependent antitumor effects. The precise structure and specific active sites of the nanoclusters provide support for exploring the interaction between chirality and the interface between biomaterials and organisms, and also pave the way for the early diagnosis and personalized treatment of diseases caused by changes in biothiols in vivo.

[0108] The applicant declares that, in the process of describing the above-mentioned specification:

[0109] The terms "this embodiment," "an embodiment of the present invention," "as shown," "further," and "further improved technical solutions," etc., indicate that the specific features, structures, materials, or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example, and the specific features, structures, materials, or characteristics described can be combined or combined in any suitable manner in one or more embodiments or examples. Furthermore, without causing contradiction, those skilled in the art can combine or combine the different embodiments or examples described in this specification and the features of the different embodiments or examples.

[0110] Finally, it should be noted that:

[0111] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them;

[0112] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Non-essential improvements, adjustments or substitutions made by those skilled in the art based on the content of this specification are all within the scope of protection claimed by the present invention.

Claims

1. A method for preparing chiral PEG-Ag6D / LSP6 cluster-based nanoparticles, characterized in that, Includes the following steps: S1. Preparation of chiral nanoclusters: Silver salt and ligands of thiazolidinthone were added to a mixed solvent, and the mixture was volatilized to obtain crystals of chiral silver nanoclusters. S2. A certain amount of the amphiphilic polymer DSPE-PEG2000 and the chiral silver nanoclusters obtained in step S1 are dissolved in an organic solvent, and then added to a certain volume of ultrapure water to prepare water-soluble chiral cluster-based nanoparticles through self-assembly; the initial concentration of the chiral silver nanoclusters is 0.2-0.4 mg / mL; the mass ratio of the silver nanoclusters to the amphiphilic polymer DSPE-PEG2000 is 1:(1-3); the organic solvent includes THF and CH2Cl2, and the volume ratio of the organic solvent to ultrapure water is 1:(5-20). S3. The reaction was carried out under vigorous stirring until the organic solvent was completely removed. The reaction was then stopped, and the product was filtered to obtain biocompatible chiral PEG-Ag6D / LSP6 cluster-based nanoparticles.

2. The method for preparing chiral PEG-Ag6D / LSP6 cluster-based nanoparticles as described in claim 1, characterized in that, In step S1, the silver salt is selected from one or more combinations of AgNO3, AgBF4, AgF6P and CF3SO3Ag, and the mixed solvent is selected from multiple combinations of DMF, DMA, DMSO, CH3CN, CH2Cl2, n-hexane and THF.

3. A chiral PEG-Ag6D / LSP6 cluster-based nanoparticle, characterized in that, Prepared by the preparation method according to any one of claims 1 to 2, the chiral PEG-Ag6D / LSP6 cluster-based nanoparticles have a spherical or ellipsoidal morphology and a particle size of 80 nm–110 nm as shown in the transmission electron microscope image.

4. The application of the chiral PEG-Ag6D / LSP6 cluster-based nanoparticles prepared by the method according to any one of claims 1-2, or the chiral PEG-Ag6D / LSP6 cluster-based nanoparticles according to claim 3, in reagents for distinguishing biothiols, enantioselective chiral recognition, or quantitative detection of biothiols in plasma, characterized in that, Includes the following steps: R1. Dissolve chiral PEG-Ag6D / LSP6 cluster-based nanoparticles in solvent A to prepare a stock solution of a specific concentration, and then dilute it to an appropriate concentration for later use. R2. Take the sample to be tested, add the diluent prepared in step R1, and then determine the type and content of biothiols in the sample based on the presence and change of the CD signal.

5. The application as described in claim 4, characterized in that, Solvent A includes ultrapure water or phosphate buffer.

6. The use of the chiral PEG-Ag6D / LSP6 cluster-based nanoparticles prepared by the method according to any one of claims 1-2, or the chiral PEG-Ag6D / LSP6 cluster-based nanoparticles according to claim 3, in the preparation of chiral-dependent antitumor drugs, characterized in that, The chiral PEG-Ag6D / LSP6 cluster-based nanoparticles are of the D configuration.

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