A hypoxia response-based platinum ultra-nanoparticle urine detection method and application

Abstract

The application discloses a urine detection method based on platinum ultra-nanoparticles responding to hypoxia and application thereof. The platinum ultra-nanoparticles PNCA are formed by covalently connecting the ultra-small platinum nanoclusters after amination with 4,4'-dicarboxylic azobenzene azo responding to hypoxia through EDC / NHS reaction, and after wrapping the nanoparticles with a layer of hyaluronic acid HA, the nanoparticles can specifically target tumor sites. Then, under the action of the hypoxia environment reductase in the tumor site, the PNCA nanoparticles can be reduced and degraded into many small PtNCs, re-entering the blood circulation, and since the particle size is less than the critical value 5.5 nm that can be removed by the kidney, the nanoparticles can be excreted from the kidney to the urine for disease state detection. Due to the excellent hypoxia response effect of the PNCA nanoparticles and the high kidney removal effect of the PtNCs, the application has obvious advantages in in vivo application.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a urine detection method and application based on hypoxia-responsive platinum ultrananoparticles. Background Technology

[0002] Early diagnosis of cancer allows for effective treatment of the primary tumor through local interventions such as surgery and radiotherapy. Current early cancer diagnosis strategies primarily rely on imaging techniques and molecular diagnostic analyses. Imaging techniques, such as magnetic resonance imaging (MRI) and computed tomography (CT), can provide high spatial and density resolution three-dimensional images and remain the standard for clinical cancer detection. However, these techniques are expensive and have relatively low sensitivity. Molecular diagnostics is currently the main method for early cancer diagnosis, but the low shedding rate of biomarkers at tumor sites and significant dilution through blood circulation limit the sensitivity of strategies for detecting circulating endogenous biomarkers due to inherent sensitivity limitations in early-stage diseases. Rather than searching for endogenous biomarkers, a promising strategy is to design exogenous biomarkers that can overcome the insufficient abundance of endogenous biomarkers and specifically detect the presence of diseased tissue. Combining platinum ultrananoparticles as exogenous biomarkers with non-invasive urine testing methods for early disease detection is a promising strategy. Summary of the Invention

[0003] To construct a platform for tumor detection using platinum ultrananoparticles based on hypoxia response, this invention connects platinum nanoclusters (PtNCs, 1-2 nm) with carboxyl groups on EDC / NHS-activated 4,4'-dicarboxylic acid azobenzene (azo) to form platinum ultrananoparticles (PNCA). In the hypoxic environment of a tumor, the N=N bonds are broken, and PNCA dissociates, releasing the ultrasmall PtNCs into the bloodstream. These PtNCs are then metabolized by the kidneys and excreted in the urine. By collecting urine and detecting the PtNC content, the disease status can be determined.

[0004] To achieve the above-mentioned objectives, the present invention relates to a scheme for the use of hypoxia-responsive platinum ultrananoparticles in urine detection of tumors, as follows: A method for preparing hypoxia-responsive platinum ultrananoparticles according to the present invention includes the following steps:

[0005] (1) The surface of ultra-small platinum nanoclusters PtNCs is aminated, and then platinum nanoclusters PtNCs are covalently linked by 4,4'-dicarboxylic acid azobenzene azo with hypoxia response to obtain platinum ultra-nanoparticles PNCA. The carboxyl group on 4,4'-dicarboxylic acid azobenzene azo is covalently linked to PtNCs after being activated by EDC / NHS reaction.

[0006] (2) Under the action of electrostatic adsorption, hyaluronic acid HA, which can specifically target the CD44 receptor on the surface of tumor cells, is wrapped on the outermost layer of PNCA nanoparticles to prepare PNCAH platinum super nanoparticles.

[0007] Further, in step (1), the platinum nanoclusters PtNCs are synthesized by a one-pot thermal method using chloroplatinic acid hexahydrate and trisodium citrate. Trisodium citrate acts as a reducing agent and a protecting agent in the reaction. The concentration of chloroplatinic acid hexahydrate used in the reaction is 1 mM, the concentration of trisodium citrate is 3.88 mM, and the reaction temperature is 100°C.

[0008] Furthermore, in step (1), the molecule used in the amination process is mercaptoethylamine, and mercaptoethanol is also added during the amination process to prevent the aggregation of platinum nanoclusters. The concentration of mercaptoethylamine during the amination reaction is 0.4 mM, the concentration of mercaptoethanol is 0.6 mM, and the reaction time is 3 h.

[0009] Furthermore, in step (1), the EDC / NHS activation process, where EDC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and NHS is N-hydroxysuccinimide, has a concentration of 30 mM for EDC and 3.3 mM for NHS, and an activation time of 2 h; the mass ratio of platinum nanoclusters PtNCs to 4,4'-dicarboxylic acid azobenzene azo is 90-100:1-6, and the reaction time is 12 h.

[0010] Furthermore, in step (2), during the HA encapsulation process, the mass ratio of HA to the added PNCA nanoparticles is 1:2, and the reaction time is 2 hours.

[0011] The urine detection method based on hypoxia-responsive platinum supernanoparticles of the present invention includes the following steps: when platinum supernanoparticles with targeting effect are delivered into cells or mice, they can dissociate into many small PtNCs in the tumor site under hypoxia, re-enter the blood circulation, and then be excreted into the urine through the kidneys for tumor status detection.

[0012] Furthermore, in the detection process described above, the N=N bonds are broken in the hypoxic environment of the tumor, and the PNCA nanoparticles dissociate to release PtNCs. The optimal renal metabolism time of PtNCs in vivo is 4 hours. Therefore, this invention selects 4 hours as the optimal detection time.

[0013] Furthermore, by utilizing the excellent catalase activity of PtNCs, it is possible to catalyze the production of oxygen from hydrogen peroxide for detection, thereby enabling the reading of disease states.

[0014] Furthermore, the cells used were MDA-MB-231 triple-negative breast cancer cells, and the mice were BALB / c nude mice. When the platinum super nanoparticles were delivered into the cells or mice, the dissociation of nanomaterials at the tumor site and the diagnosis of tumor status were achieved.

[0015] The application of the platinum supernanoparticles PNCAH described in this invention as exogenous biomarkers.

[0016] Compared with the prior art, the beneficial effects of the present invention are: (1) The present invention uses exogenous biomarkers to replace low-abundance endogenous biomarkers for detection, which can effectively detect disease states and improve the sensitivity of tumor detection; (2) Due to the excellent hypoxia dissociation performance of PNCA nanoparticles and the renal metabolic effect of PtNCs, the present invention can be well applied to living organisms. Attached Figure Description

[0017] Figure 1 (A) is a schematic diagram of the reaction principle of the method; (B) is a method for preparing PNCAH nanoparticles; (C) is a schematic diagram of in vivo tumor urine detection of hypoxia-responsive PNCAH nanoparticles.

[0018] Figure 2 These are the potentials for each process of nanoparticle modification; the potentials from left to right in the figure are PtNCs, PtNCs-NH2 / OH, PNCA, and PNCAH.

[0019] Figure 3 This describes the oxygen-deficient dissociation of PNCA nanoparticles. Figure 3 A is a fluorescence intensity graph showing the change of PNCA nanoparticles over time after treatment with the same concentration of SDT (20 mM). Figure 3 B is the fluorescence spectrum of PNCA nanoparticles after being treated with different concentrations of SDT for the same time (50 min).

[0020] Figure 4 The activity of MDA-MB-231 cells in the presence of PNCAH nanoparticles was measured by the CCK-8 assay.

[0021] Figure 5 This shows the metabolism of PNCAH nanoparticles in triple-negative breast cancer (TNBC) mice and healthy mice at different times.

[0022] Figure 6 This is a graph showing the test results of triple-negative breast cancer (TNBC) mice and healthy mice. Detailed Implementation

[0023] To better understand the present invention, the following embodiments and accompanying drawings further illustrate the content of the invention, but the content of the invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods used are conventional methods.

[0024] Example 1

[0025] This embodiment provides a method for preparing PNCAH nanoparticles:

[0026] PtNCs (0.01 g) were amination with mercaptoethylamine (SH-NH2, 0.4 mM) and mercaptoethanol (SH-OH, 0.6 mM). The main function of mercaptoethanol was to prevent PtNCs from agglomerating. The reaction was carried out for 3 h. Then, 0.002 g of azo was placed in 200 μL of dimethyl sulfoxide (DMSO) and activated with carboxyl groups using 0.038 g of N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide (EDC) and 0.0058 g of N-hydroxysuccinimide (NHS). The reaction was carried out for 2 h. The activated azo solution was then added to the amination PtNCs under magnetic stirring. The solvent was 10 mM pH 7.4 PBS. The stirring speed was 480 rpm. After reacting for 12 h, the mixture was washed three times with deionized water, and the PNCA nanoparticles were collected and transferred to 5 mL of deionized water. Then, hyaluronic acid (HA, 5 mg) was added to the PNCA solution and reacted for 2 h with magnetic stirring at 550 rpm. After centrifugation and washing three times with deionized water, PNCAH nanoparticles were obtained and further equilibrated at room temperature for 5 min before use in cell and animal studies. The surface modification in this process can be verified using potential analysis. Figure 2 ).

[0027] Example 2

[0028] Oxygen-deficient dissociation of PNCAH nanoparticles

[0029] This embodiment verifies the hypoxia response of the system, as follows:

[0030] First, PtNCs were labeled with Cy5 fluorescent dye. Then, the labeled PtNCs were assembled into PNCA nanoparticles. An oxygen-deprivation mimic, SDT (20 mM), was added, and the reaction was carried out with shaking at room temperature. The fluorescence intensity at 670 nm (Cy5 emission wavelength) was measured every 10 minutes. The results are as follows: Figure 3 As shown in Figure A, the fluorescence intensity continuously increases with time, indicating that the PNCA nanoparticles have dissociated; simultaneously, the fluorescence intensity also continuously increases with increasing SDT concentration, which further verifies the dissociation of the PNCA nanoparticles. Figure 3 B).

[0031] Example 3

[0032] PNCAH cytotoxicity

[0033] The cytotoxicity of the PNCAH nanoparticles in Example 1 was tested using the following method:

[0034] Triple-negative breast cancer cells (MDA-MB-231 cells) were selected, and different concentrations of PNCAH nanoparticles were used for incubation. It was found that the nanoparticles showed no significant toxicity to the cells below 10 μg / mL. Figure 4 To reduce the toxic effects of PNCAH nanoparticles on cells, the concentration of PNCAH nanoparticles was controlled at 10 μg / mL in subsequent cell experiments.

[0035] Example 4

[0036] Determination of in vivo detection time

[0037] The optimal time for in vivo detection of this system was determined by detecting the metabolism of PNCAH nanoparticles in vivo at different times.

[0038] This embodiment provides a study on the metabolic time of PNCAH nanoparticles in vivo: A nanocomposite targeting tumor sites was prepared according to Example 1. Two groups of mice were selected, five mice in each group: one group consisted of triple-negative breast cancer mice, and the other group consisted of healthy mice. PNCAH nanoparticles were then injected into the triple-negative breast cancer mice and healthy mice via the tail vein, respectively. Urine was collected every 2 hours, and the PtNCs content in the urine was measured. Figure 5 As shown, the detection signal in triple-negative breast cancer mice gradually increased from 0 to 4 hours, then gradually decreased with time, while the detection signal in healthy mice showed no significant change throughout the process. Furthermore, this invention demonstrates a very significant difference in the detection signals between triple-negative breast cancer mice and healthy mice within 0-4 hours, peaking at 4 hours. Considering both the shortest detection time and the optimal detection signal, 4 hours was chosen as the optimal detection time.

[0039] Example 5

[0040] The system's effectiveness in detecting tumors in mice.

[0041] All animal experiments in this embodiment were conducted under the guidance of the Experimental Animal Use and Management Committee of Nanjing University. This embodiment verifies the detection effect of hypoxia-responsive PNCAH nanoparticles in vivo in mice. Specific experimental steps are as follows:

[0042] Thirty BALB / c mice were divided into two groups of 15 each. One group of mice underwent tumor grafting (MDA-MB-231 cells). After tumor growth, both groups were injected with equal amounts of PNCAH nanoparticles (2.0 mg / kg) to assess disease status. Urine samples were collected from the mice within 4 hours for analysis. Results are as follows: Figure 6 The detection results in triple-negative breast cancer mice were significantly higher than those in healthy mice, showing a statistically significant difference (P<0.0001). This means that the use of hypoxia-responsive platinum ultrananoparticles in urine for tumor detection according to this invention can effectively distinguish between tumor-bearing mice and healthy mice, proving the successful completion of tumor detection.

[0043] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing platinum ultra-nanoparticles based on hypoxia response, characterized by The preparation method comprises the following steps: (1) amino treatment is performed on the surface of ultra-small platinum nanoclusters PtNCs, the particle size of the platinum nanoclusters PtNCs is 1-2 nm, and the amino treatment is specifically performed by adding mercaptoethylamine for amino reaction and adding mercaptoethanol to prevent aggregation, and the reaction time is 3 hours; (2) after the carboxyl of 4,4'-dicarboxylic acid azobenzene azo is activated through an EDC / NHS reaction, the activated carboxyl is covalently connected with the amino-treated platinum nanoclusters PtNCs, and the PNCA nanoparticles are collected; the mass ratio of the platinum nanoclusters PtNCs to the 4,4'-dicarboxylic acid azobenzene azo is 90-100:1-6, the reaction time is 12 hours, the concentration of EDC in the reaction is 30 mM, the concentration of NHS in the reaction is 3.3 mM, and the activation time is 2 h; (3) under the action of electrostatic adsorption, hyaluronic acid HA that is specific to the CD44 receptor on the surface of tumor cells is wrapped on the outermost layer of the obtained PNCA nanoparticles, the mass ratio of the HA to the added PNCA nanoparticles in the wrapping process is 1:2, the wrapping time is 2 hours, and the platinum ultra-nanoparticles based on hypoxia response are prepared.

2. The method for preparing platinum ultrananoparticles based on hypoxia response according to claim 1, characterized in that: In step (1), the platinum nanoclusters PtNCs are synthesized by one-pot hot synthesis using chloroplatinic acid hexahydrate and trisodium citrate, wherein the concentration of the chloroplatinic acid hexahydrate is 1 mM, the concentration of the trisodium citrate is 3.88 mM, and the reaction temperature is 100 DEG C.

3. The method for preparing platinum ultrananoparticles based on hypoxia response according to claim 2, characterized in that: In step (1), the concentration of the mercaptoethylamine is 0.4 mM, the concentration of the mercaptoethanol is 0.6 mM, and the reaction time is 3 h.

4. Use of the hypoxia-responsive platinum ultra-nanoparticles prepared by the method according to any one of claims 1 to 3 for the preparation of a product for detecting the state of a tumor, characterized in that The preparation method comprises the following steps: (1) the platinum ultra-nanoparticles are prepared by using the preparation method in any one of claims 1 to 3; (2) the prepared platinum ultra-nanoparticles are delivered into cells or mice; (3) in a tumor hypoxic environment, the platinum ultra-nanoparticles are dissociated into small platinum nanoclusters PtNCs, and the platinum nanoclusters PtNCs are excreted into urine through the kidneys; (4) urine samples are collected, and the presence of a tumor state is determined by detecting the content of the platinum nanoclusters PtNCs in the urine.

5. The hypoxia-responsive platinum ultra-nanoparticle-based urine test method according to claim 4, characterized by: In the detection process, in a tumor hypoxic environment, the N=N bond is broken, the platinum ultra-nanoparticles are dissociated to release PtNCs, and the optimal kidney metabolism time of the PtNCs in the body is 4 hours.

6. The hypoxia-responsive platinum ultra-nanoparticle-based urine test method according to claim 5, characterized by: The catalysis of the peroxidase activity of the PtNCs is used to generate oxygen by catalyzing hydrogen peroxide for detection.

7. The hypoxia-responsive platinum ultra-nanoparticle-based urine test method according to claim 5, characterized by: The cells are MDA-MB-231 triple-negative breast cancer cells, and the mice are BALB / c nude mice.

8. The platinum ultra-nanoparticles prepared by the preparation method in claim 1 are used as exogenous biomarkers in the preparation of a product for detecting a tumor state.

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