Organic nitroxide free radical nano-composite as well as preparation method and application thereof

Abstract

The invention relates to a preparation method and application of an organic nitroxide free radical nanocomposite, and belongs to the technical field of biomedical materials. The organic nitroxide free radical nanocomposite comprises a micelle formed on the basis of a PLGA carrier, and TEMP bonded with the micelle through an amido bond. The organic nitroxide free radical nanocomposite has active oxygen responsive magnetic resonance imaging activity. Contrast of a tumor area can be increased, and boundaries of tumor tissues and normal tissues can be distinguished; and the quantitative detection of active oxygen can be realized. The method is expected to break through the dependence limitation of a traditional imaging technology on an anatomical structure, accurately distinguish normal tissue and tumor tissue boundaries, realize boundary definition of a molecular level, and bring new breakthrough and hope for early diagnosis of cancers.

Description

Technical Field

[0001] The invention relates to an organic nitroxide free radical nanocomposite and a preparation method and application thereof, belonging to the technical field of biomedical materials. Background Art

[0002] The latest cancer report shows that in 2022, there will be 18.74 million new cancer cases and 9.67 million cancer deaths worldwide, of which my country accounts for 24%. In the field of cancer imaging diagnosis, methods such as ultrasound imaging, X-ray imaging, and magnetic resonance imaging (MRI) are widely used. Among them, MRI, with its advantages of high resolution and multi-parameter imaging, can clearly show the morphology, size, boundaries, and relationship of cancer to surrounding tissues. It effectively compensates for the shortcomings of ultrasound in poor visualization of deep lesions and X-ray radiation, providing key support for early cancer diagnosis. However, because the imaging features of cancer are not obvious in the early stages, it is often necessary to wait until more significant imaging features develop before combining multiple imaging technologies to confirm the diagnosis. However, because the imaging features of cancer are not obvious in the early stages, but cancer cells develop extremely rapidly, this leads to a "window period" between the early stages of cancer and diagnosis, causing patients to miss the optimal treatment opportunity. The primary tumor is often already in the advanced stage when it is diagnosed.

[0003] In recent years, with the deepening of research on the tumor microenvironment, people have gradually realized that changes in the tumor microenvironment are often related to the malignancy of the tumor, and play a vital role in the occurrence, development, metastasis and response to treatment of the tumor. Compared with normal tissue, tumor tissue has unique characteristics such as lower acidity and higher concentration of reactive oxygen species. Among them, reactive oxygen species, as a key signaling molecule, has a significantly higher concentration in tumor cells than in normal tissues, and the concentration ratio between the two can reach 10 to 100 times, which provides a specific target for early and accurate imaging. However, due to the transient nature and complexity of the biological environment, measuring reactive oxygen species in vivo is challenging. Summary of the Invention

[0004] In order to solve the above problems existing in the prior art, the purpose of the present invention is to provide an organic nitroxide free radical nanocomposite and a preparation method and application thereof, so as to overcome the shortcomings of the prior art.

[0005] The first object of the present invention is achieved through the following technical solutions:

[0006] The invention discloses an organic nitroxide free radical nanocomposite, which comprises a micelle formed based on a PLGA carrier and 2,2,6,6-tetramethylpiperidine (TEMP) bonded to the micelle via an amide bond.

[0007] Preferably, the PLGA carrier is one or more of PLGA-COOH, HOOC-PLGA-PEG, and PLGA-PEG-COOH; more preferably, the PLGA carrier is PLGA-PEG-COOH.

[0008] Preferably, the average particle size of the organic nitroxide free radical nanocomposite is 2 to 50 nm, more preferably 5 to 20 nm, and even more preferably 10 to 15 nm.

[0009] Preferably, the organic nitroxide free radical nanocomposite generates paramagnetism under the action of active oxygen and shows a triplet peak in electron spin resonance (ESR).

[0010] Preferably, the organic nitroxide nanocomposite is responsive to the tumor microenvironment, which includes reactive oxygen species, an acidic environment, etc. Further preferably, the organic nitroxide nanocomposite is active in magnetic resonance imaging responsive to reactive oxygen species.

[0011] Preferably, the organic nitroxide free radical nanocomposite increases the contrast of the tumor area and distinguishes the boundary between tumor tissue and normal tissue.

[0012] Preferably, the organic nitroxide nanocomposite can be used for quantitative detection of reactive oxygen species with a detection limit of 20 to 50 uM.

[0013] The second object of the present invention is achieved through the following technical solutions:

[0014] A method for preparing an organic nitroxide free radical nanocomposite comprises the following steps:

[0015] (1) dissolving the PLGA carrier in an organic solvent, then dripping it into water, and removing the organic solvent to obtain a nanoparticle carrier solution;

[0016] (2) The nanoparticle carrier solution and TEMP-NH2 are stirred and reacted in the presence of an amidation reaction auxiliary agent, and the nanoparticles are coupled to TEMP through an amide bond;

[0017] (3) The reaction solution of step (2) is dialyzed and freeze-dried to obtain an organic nitroxide free radical nanocomposite.

[0018] Preferably, the volume ratio of the organic solvent to the water is 1:1 to 1:10; for example, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, but is not limited to the values ​​listed above, and other values ​​not listed within the numerical range are also applicable. More preferably, the volume ratio of the organic solvent to the water is 1:2 to 1:5.

[0019] Preferably, the organic solvent includes but is not limited to one or more of acetone, anhydrous ethanol, dichloromethane, chloroform, etc. More preferably, the solvent is acetone.

[0020] Preferably, the organic solvent is removed by a solvent volatilization method, which comprises stirring at 100-1000 rpm / min for 5-24 hours at room temperature. More preferably, stirring at 300-800 rpm / min for 8-20 hours.

[0021] Preferably, the molar ratio of TEMP-NH2 to carboxyl groups in the nanoparticle carrier solution is 1:0.3-3.

[0022] The molar amount of the amidation reaction auxiliary agent is added based on the amount of auxiliary agent normally required for amidation between the amino group and the carboxyl group.

[0023] Preferably, TEMP-NH2 is a combination of one or more of 3-amino-2,2,6,6-tetramethylpiperidine, 4-amino-2,2,6,6-tetramethylpiperidine, and 5-amino-2,2,6,6-tetramethylpiperidine.

[0024] Preferably, the amidation reaction auxiliary agent is one or more of: EDC·HCl / NHS, DCC / NHS, EDC·HCl / HOBt, and DIC / HOBt.

[0025] Preferably, the stirring reaction temperature in step (2) is 10-40° C., and the reaction time is 10-40 h.

[0026] Preferably, the molecular weight cut-off of the dialysis bag used for dialysis is 7000 to 20000, and the dialysis time is 12 to 100 hours.

[0027] The third object of the present invention is achieved through the following technical solutions:

[0028] Use of an organic nitroxide free radical nanocomposite in any of the following a)-e):

[0029] a) preparing a magnetic resonance imaging contrast agent;

[0030] b) preparing a medicament for use in magnetic resonance imaging diagnosis of cancer;

[0031] c) preparing a medicament for alleviating symptoms in cancer patients;

[0032] d) preparing a drug for treating cancer;

[0033] e) Quantitative detection of reactive oxygen species not for therapeutic or diagnostic purposes.

[0034] The cancer includes one or more of lung cancer, breast cancer, liver cancer, gastric cancer, colorectal cancer, prostate cancer, ovarian cancer, brain cancer, etc.

[0035] Preferably, the use of the organic nitroxide nanocomposite for quantitative detection of reactive oxygen species not for therapeutic or diagnostic purposes comprises the following steps:

[0036] 1) Preparation of standard curve

[0037] H2O2 solutions with different concentration gradients, including an H2O2 solution with a concentration of 0, were prepared, and organic nitroxide free radical nanocomposite solutions were added thereto for incubation. T1-weighted MR scanning was then performed using a nuclear magnetic resonance imaging device. Signal values ​​were extracted from the original images to obtain a standard curve with the H2O2 solution concentration on the abscissa and the signal intensity on the ordinate, and the standard curve equation was obtained.

[0038] 2) The organic nitroxide free radical nanocomplex is injected into the body, and a T1-weighted MR scan is performed using a nuclear magnetic resonance imaging device. Signal values ​​are extracted from the original image, and the reactive oxygen species concentration is determined according to the signal value intensity using a standard curve equation.

[0039] Preferably, the incubation time is 1 to 6 hours.

[0040] Preferably, the detection limit is calculated to be 20 to 50 uM according to the standard curve.

[0041] Quantitative detection of reactive oxygen species can be used for drug development, treatment efficacy evaluation, health management, and distinguishing tumor boundaries. Drug development: For example, detecting the concentration of reactive oxygen species in the body can be used to screen out anti-cancer drugs that are sensitive to ROS. Treatment efficacy evaluation: For example, predicting the probability of postoperative metastasis by changes in ROS concentration in the body. Health management: Predicting disease risks by detecting changes in ROS concentration in the body, suggesting adjustments to exercise intensity, work and rest time, eating habits, etc. to avoid the occurrence of diseases. Tumor diagnosis: As a key signaling molecule, the concentration of reactive oxygen species in tumor cells is significantly higher than that in normal tissues. By monitoring the ROS concentration in the body to distinguish the boundary between tumor tissue and normal tissue, a quantifiable indicator is provided for the definition of tumor boundaries.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The organic nitroxide free radical nanocomposite prepared by the present invention has a small average particle size, has abundant carboxyl functional groups on the surface, can be well dispersed in deionized water, and is easy to surface modify and biologically apply.

[0044] 2. The organic nitroxide free radical nanocomposite prepared by the present invention has the activity of active oxygen responsive magnetic resonance imaging, greatly improves the contrast in the tumor area, sensitively distinguishes the boundary between tumor tissue and normal tissue, and can be used for cancer diagnosis.

[0045] 3. The organic nitroxide free radical nanocomplex prepared by the present invention can be used as a drug delivery system for cancer therapeutic drugs. It has excellent tumor microenvironment acid response ability and can accurately deliver drugs to cancer cells, realizing a pH-responsive nanodrug delivery system, and ultimately achieving MRI-guided efficient cancer treatment.

[0046] 4. The organic nitroxide free radical nanocomposite prepared by the present invention can quantitatively detect reactive oxygen species, and the detection limit of reactive oxygen species is as low as 27.04 μM.

[0047] 5. The organic nitroxide free radical nanocomplex prepared by this invention significantly enhances T1-weighted MRI signals through the paramagnetic relaxation effect. This allows for real-time mapping of tumor invasion boundaries via reactive oxygen species concentration gradients, capturing metabolic abnormalities before the tumor undergoes significant morphological changes, thus advancing the diagnostic window. This novel nanoprobe is expected to transcend the limitations of traditional imaging techniques that rely on anatomical structures, accurately distinguishing the boundaries between normal and tumor tissues and achieving molecular-level boundary definition, bringing new breakthroughs and hope to early cancer diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 TEM image of CPPT NPs obtained in Example 1.

[0049] Figure 2 The ESR spectra of CPPT NPs obtained in Example 1 in the presence and absence of hydrogen peroxide are shown.

[0050] Figure 3 The ESR spectra of the CPPT NPs obtained in Example 1 in the presence and absence of singlet oxygen are shown.

[0051] Figure 4 The magnetic resonance imaging performance diagram of the CPPT NPs in response to hydrogen peroxide obtained in Example 1 is shown, wherein A is the T1-weighted MR imaging image of CPPT NPs in response to different H2O2 concentrations, B is the quantitative signal intensity image reconstructed from the T1-weighted MR imaging image, and C is the color image reconstructed from the T1-weighted MR imaging image; D is the comparison of the r1 relaxation rate of CPPT NPs in response to different H2O2 concentrations.

[0052] Figure 5 The magnetic resonance imaging performance of CPPT NPs singlet oxygen response obtained in Example 1 is shown, wherein A represents the different responses of CPPT NPs. 1Figure 3 is a T1-weighted MR imaging image of O2 concentration. B is a color image reconstructed from the T1-weighted MR imaging image. C is a quantitative signal intensity image reconstructed from the T1-weighted MR imaging image. In Figure C, - represents ultrasound time 0 min, + represents ultrasound time 2 min, ++ represents ultrasound time 5 min, and +++ represents ultrasound time 10 min.

[0053] Figure 6 The T1-weighted MR imaging images and standard curves of the CPPT NPs obtained in Example 1 in response to different concentrations of hydrogen peroxide are shown; wherein A is the T1-weighted MR imaging image of the CPPT NPs in response to different H2O2 concentrations, and B is the linear regression equation and detection limit of the signal intensity extracted from the T1-weighted MR imaging image and the H2O2 concentration.

[0054] Figure 7 The magnetic resonance imaging performance diagrams of the CPPT NPs obtained in Example 1 in normal cells and tumor cells are shown, wherein A is the T1-weighted MR imaging image of CPPT NPs under different treatment methods, + / - represents the presence / absence of this component, and B is the quantitative signal intensity image reconstructed from the T1-weighted MR imaging image. DETAILED DESCRIPTION

[0055] Below by specific embodiment and accompanying drawing, technical scheme of the present invention is further described explanation, it should be understood that specific embodiment described herein is only for helping to understand the present invention, is not used for specific limitation of the present invention.And accompanying drawing used herein, is only for better illustrating the disclosure of the present invention, does not have limiting effect on protection scope.If no special instructions, the raw materials adopted in the embodiment of the present invention are all raw materials commonly used in this area, and the method adopted in the embodiment is all conventional method in this area.

[0056] It should be noted that, in this article, "particle size" refers to the maximum distance between any two points on the particle outline observed using an SEM, TEM, or other observation equipment. The "average particle size" value is calculated as the average particle size of particles observed in dozens of fields of view using an SEM, TEM, or other observation equipment.

[0057] In the following examples, PLGA-PEG-COOH was purchased from Xi'an Haoran Biotechnology Co., Ltd., with a molecular weight of 18,400 and a carboxyl content of ≥25%.

[0058] Example 1

[0059] The organic nitroxide free radical nanocomposite of this embodiment is obtained by the following preparation method:

[0060] S1. Add 50 g of PLGA-PEG-COOH into 10 ml of acetone to fully dissolve the raw materials to obtain a precursor solution;

[0061] S2. Add the precursor solution dropwise into deionized water with a volume ratio of acetone to deionized water of 1:4, add a stirring bar, and stir at a speed of 600 rpm / min in an air atmosphere for 12 h to remove the organic solvent acetone, thereby obtaining a nanoparticle carrier solution;

[0062] S4. Add 1 mmol of 4-amino-2,2,6,6-tetramethylpiperidine, 1.4 mmol of EDC·HCl, and 1.4 mmol of NHS to the nanoparticle carrier solution, and stir at room temperature (25° C.) and 600 rpm / min for 24 h to obtain an organic nitroxide free radical nanocomposite mixed solution;

[0063] S3. The organic nitroxide free radical nanocomposite mixed solution was taken and placed into a dialysis bag with a molecular weight cutoff of 14,000. The solution was dialyzed in ultrapure water for 72 hours and then freeze-dried for 72 hours to obtain a reddish-brown nanocomposite (abbreviated as: CPPTNPs).

[0064] Figure 1 This is a TEM image of the organic nitroxide free radical nanocomposite obtained in Example 1. The results show that the nanoparticles are nearly round particles with an average particle size of about 15 nm.

[0065] Example 2

[0066] The organic nitroxide free radical nanocomposite of this embodiment is obtained by the following preparation method:

[0067] S1. Add 50 g of PLGA-PEG-COOH into 10 ml of acetone to fully dissolve the raw materials to obtain a precursor solution;

[0068] S2. Add the precursor solution dropwise into deionized water with a volume ratio of acetone to deionized water of 1:6, add a stirring bar, and stir at a speed of 400 rpm / min under air atmosphere for 10 h to remove the organic solvent acetone, thereby obtaining a nanoparticle carrier solution;

[0069] S4. Add 1.2 mmol 4-amino-2,2,6,6-tetramethylpiperidine, 1.5 mmol EDC·HCl, and 1.5 mmol NHS to the nanoparticle carrier solution, and stir at room temperature (25°C) and 500 rpm / min for 20 h to obtain an organic nitroxide free radical nanocomposite mixed solution;

[0070] S3. The organic nitroxide free radical nanocomposite mixed solution was taken and placed into a dialysis bag with a molecular weight cutoff of 14,000. The solution was dialyzed in ultrapure water for 60 hours and then freeze-dried for 60 hours to finally obtain a reddish-brown nanocomposite.

[0071] 1. ESR test

[0072] The organic nitroxide free radical nanocomposite of Example 1 was used to prepare a 1 mg / ml aqueous solution of CPPT NPs. After incubation for 4 h in the presence or absence of H2O2 (200 μM), the results were compared with a 1 mg / ml aqueous solution of 2,2,6,6-tetramethylpiperidinium nitroxide (TEMPO) and tested on an electron paramagnetic resonance (ESR) spectrometer. The ESR results showed that the organic nitroxide free radical nanocomposite produced a characteristic triplet peak of TEMPO ( Figure 2 ), demonstrating that CPPT NPs can be transformed into paramagnetic TEMPO in response to H2O2, and are expected to be used in the development of T1 contrast agents.

[0073] A certain amount of chlorin E6 (Ce6) was weighed and dissolved in methanol to a final concentration of 1 mM. A 1 mg / ml CPPT NPs aqueous solution and Ce6 solution were mixed and then subjected to ESR under the presence or absence of ultrasonic power stimulation (0.5 W / cm 2 , 2min). The photosensitizer Ce6 is excited to produce singlet oxygen ( 1 O2), reacts with the organic nitroxide free radical nanocomplex to produce the characteristic triplet peak of TEMPO, indicating that the contrast agent can successfully respond 1 O2 and produces paramagnetism ( Figure 3 ), which fully demonstrates that organic nitroxide nanocomposites have the ability to respond to ROS and be applied in MR imaging.

[0074] 2. ROS Response MRI Performance Test

[0075] 2.1 Hydrogen peroxide-responsive magnetic resonance imaging performance

[0076] Different concentrations (0, 125, 250, 500, 1000, 2000, and 4000 μg / mL) of organic nitroxide nanocomposite aqueous solutions were prepared, and 4 mL was added to seven centrifuge tubes as a group, for a total of 28 samples in four groups. Then, different concentrations (0, 50, 100, and 200 μM) of H2O2 solution were added to each group of samples, and the samples were left to stand for 4 hours before being tested. All samples were uniformly scanned with a clinical 3.0T magnetic resonance imaging device for T1-weighted MR scanning, and longitudinal relaxation time was measured to calculate longitudinal relaxation rate (r1). Scan parameters were set at TR = 300 ms and TE = 9.7 ms.

[0077] Results: With the gradual increase of reagent concentration and H2O2 concentration, the T1 signal of organic nitroxide free radical nanocomposite showed a trend of gradual enhancement ( Figure 4 A). Quantitative analysis of signal intensity shows that at 0 μM H2O2, the signal of CPPT NPs remains essentially unchanged. However, as the H2O2 concentration gradually increases to 200 μM H2O2, the T1 signal of CPPT NPs begins to rise sharply, showing a strong signal contrast on the T1 image ( Figure 4 BC). Then, by measuring the T1 relaxation time of CPPT NPs under different H2O2 concentrations, it was calculated that the corresponding relaxation rate (r1) increased with the increase of H2O2 concentration, which was 0.1774mM - 1 s -1 , 0.2137mM -1 s -1 and 0.3129mM -1 s -1 ( Figure 4 D), demonstrating the powerful ROS-responsive MR imaging capability of CPPT NPs.

[0078] Based on the pathological characteristics that the H2O2 concentration in the tumor microenvironment (TME) is significantly higher (0.1-1mM) than that in normal tissue (10-100μM), this study further established a physiological / pathological simulation system for comparison. When the CPPT NPs concentration was constant at 4000μg / mL, the r1 value of the 200μM H2O2 (simulated TME) group (0.3129mM -1 ·s -1 ) compared with 50μM H2O2 (simulating normal tissue) group (0.1774mM -1 ·s -1 ) increased by 76.3%. This concentration-dependent relaxation difference (Δr 1 =0.1355mM -1 ·s -1 ) can achieve high-contrast imaging of the tumor-normal tissue boundary to meet the needs of clinical tumor positioning.

[0079] 2.2 Singlet oxygen-responsive magnetic resonance imaging performance

[0080] To verify the effect of CPPT NPs on singlet oxygen ( 1 O2) response capability, this study established a 1 O2 generation system. Secondly, in order to verify the response performance of the material to singlet oxygen, Ce6 methanol solution (final concentration of 1mM) was used at 0.5W / cm 2The subjects were irradiated with ultrasound for different times (0 min, 2 min, 5 min, 10 min) at an ultrasound power of 100 nm to obtain singlet oxygen at different concentrations. T1-weighted MR scanning was performed on a clinical 3.0T magnetic resonance imaging device using the same grouping and testing conditions as above.

[0081] The experimental results showed that the T1 signal intensity of CPPT NPs showed a significant concentration-dependent change pattern, with the concentration of nanomaterials (0-4000 μg / mL) and 1 With the increase of O2 concentration, the T1 signal intensity gradually increased from the initial value of 62.8 to a peak value of 486.7, and then decreased to 281.6 ( Figure 5 AC). This biphasic response characteristic confirms the effect of CPPT NPs on 1 Specific response ability to O2. Combined with the previous response results to hydrogen peroxide (H2O2), it is shown that CPPT NPs have a broad spectrum of reactive oxygen species (ROS) response characteristics.

[0082] 3. Obtaining a standard curve for quantitative determination of reactive oxygen species

[0083] To quantitatively evaluate the response characteristics and detection sensitivity of CPPT NPs to reactive oxygen species (ROS), this study systematically analyzed the changes in T1-weighted signal intensity of CPPT NPs under stimulation with different concentrations of H₂O₂ by establishing a standard curve. The detection limit of this nanoprobe for ROS was calculated based on this analysis. First, seven 1 mg / mL CPPT NP solutions were prepared. 4 mL of each solution was then added to various concentrations of H₂O₂ (0, 10, 30, 60, 80, 120, and 160 μM) and incubated for 4 hours to obtain the test samples. All samples underwent T1-weighted MRI scanning using a clinical 3.0T magnetic resonance imaging system with scan parameters set to TR = 300 ms and TE = 9.7 ms. Signal values ​​were extracted from the original images to generate a standard curve with H₂O₂ concentration on the abscissa and signal intensity on the ordinate, from which the detection limit was calculated.

[0084] result:

[0085] Based on the linear fitting analysis of T1 signal intensity and H2O2 concentration (R 2 =0.979), the equation of the constructed standard curve is y=4.69x+134 (slope m=4.69), and its residual standard error (S y ) is 38.43. According to the IUPAC detection limit calculation formula LOD=3*S yThe nanoprobe's detection limit for ROS was calculated to be 27.04 μM, 73% lower than the typical ROS concentration threshold (≥100 μM) in the tumor microenvironment. Furthermore, imaging results showed that the T1 signal exhibited a gradient enhancement trend with H2O2 concentration, and a 10 μM H2O2 concentration difference could be discerned on a clinical 3.0T MRI system. This sensitivity meets the requirements for tumor boundary detection (ROS <100 μM in normal tissue, >100 μM in tumor tissue), providing a quantifiable molecular imaging indicator for tumor boundary delineation.

[0086] 4. Intracellular MRI Test

[0087] step:

[0088] First, L929, MCF-10A, and MCF-7 cells were cultured at 5×10 6 / mL were seeded in a 10 cm cell culture dish and cultured for 24 h to allow them to adhere. After 24 h, the cells were treated with different methods. The experimental groups were set as Ⅰ: Control (no cells or materials) group, Ⅱ: L929 + CPPT NPs group, Ⅲ: MCF-10A + CPPT NPs group, Ⅳ: MCF-7 group, Ⅴ: MCF-7 + CPPT NPs group, Ⅵ: MCF-7 + CPPT NPs + 50 μM H2O2 group, Ⅶ: MCF-7 + CPPT NPs + 100 μM H2O2 group, and Ⅷ: MCF-7 + CPPTNPs + 200 μM H2O2 group. Then, the original culture medium was discarded, and complete culture medium containing corresponding concentration of H2O2 was added for stimulation for 4 h. Then, the culture medium was discarded and 1 mg / mL CPPT NPs6mL, continue incubation for 1h, discard the material, digest the cells with trypsin and centrifuge, and finally resuspend the cells with 1ml of 0.5% hot agarose solution, put them in a 1.5mL centrifuge tube, quickly put them on ice to solidify, and perform T1-weighted MR scanning using a clinical 3T magnetic resonance imaging device, with the scanning parameters set to TR = 300ms, TE = 9.7ms.

[0089] result:

[0090] In order to evaluate the ability of CPPT NPs to generate T1 signals in response to ROS, MCF-7 breast cancer cells and two groups of normal cells were selected as research objects. The breast cancer cells were stimulated by H2O2 to produce different concentrations of ROS to observe the MR signals in the cells after CPPT NPs were taken up by the cells. According to the image results ( Figure 7Figure A) shows that CPPT NPs produce a significant T1 signal in MCF-7 cells, and the signal intensity increases significantly with increasing intracellular reactive oxygen species (ROS) levels, demonstrating the high sensitivity of CPPT NPs to changes in ROS concentration. Under 50 μM H2O2 stimulation, the intracellular T1 signal value reached 597.3, a 4.9-fold increase compared to the control group (Group I) value of 122.6. Under 200 μM H2O2 stimulation, the T1 signal value further increased to 682.4, a 5.6-fold increase compared to the control group. These results demonstrate that CPPT NPs not only efficiently respond to ROS species but also possess exceptionally high sensitivity in their response recognition capabilities.

[0091] In addition, under the stimulation of 200 μM H2O2, tumor cells showed a significant bright signal, achieving the effect of "lighting up" the tumor cells, with a signal value of 682.4. Compared with the signal values ​​of two normal cells, L929 and MCF-10A (222.5 and 378.4, respectively), the signal intensity was significantly increased by 206.7% and 80.3% ( Figure 7 B), reaching 3.0 times and 1.8 times the normal cell signal value, respectively. This result shows that CPPT NPs have the ability to accurately identify tumor cells and effectively distinguish tumor cells from normal cells, further highlighting its application potential in tumor-specific imaging.

[0092] The various aspects, embodiments, and features of the present invention should be considered in all respects as illustrative and not limiting, the scope of which is defined solely by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0093] In the preparation method of the present invention, the order of the steps is not limited to the order listed. Persons skilled in the art will appreciate that varying the order of the steps without inventive effort is within the scope of the present invention. Furthermore, two or more steps or actions may be performed simultaneously.

[0094] Finally, it should be noted that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit its implementation. Persons skilled in the art may make various modifications, additions, or substitute similar methods for the described specific embodiments. It is not necessary and impossible to provide comprehensive examples of all implementations here. However, obvious variations or modifications arising from the essential spirit of the present invention remain within the scope of protection of the present invention, and interpreting them as any additional limitations would be contrary to the spirit of the present invention.

Claims

1. An organic nitroxide free radical nanocomposite, characterized in that: The organic nitroxide free radical nanocomposite comprises micelles formed based on a PLGA carrier, and 2,2,6,6-tetramethylpiperidine bonded to the micelles via an amide bond; The organic nitroxide free radical nanocomposite can be used for quantitative detection of reactive oxygen species.

2. The organic nitroxide free radical nanocomposite according to claim 1, characterized in that: The average particle size of the organic nitroxide free radical nanocomposite is 2 to 50 nm.

3. The organic nitroxide free radical nanocomposite according to claim 1, characterized in that: The organic nitrogen oxide free radical nanocomposite generates paramagnetism under the action of active oxygen and shows a triplet peak in electron spin resonance; and / or, the organic nitroxide nanocomposite has reactive oxygen species responsive magnetic resonance imaging activity; And / or, the organic nitroxide free radical nanocomposite increases the contrast of the tumor area and distinguishes the boundary between tumor tissue and normal tissue.

4. A method for preparing an organic nitroxide free radical nanocomposite, characterized in that: The following steps are involved: (1) dissolving the PLGA carrier in an organic solvent, then dripping it into water, and removing the organic solvent to obtain a nanoparticle carrier solution; (2) The nanoparticle carrier solution and TEMP-NH2 are stirred and reacted in the presence of an amidation reaction auxiliary agent, and the nanoparticles are coupled to TEMP through an amide bond; (3) The reaction solution of step (2) is dialyzed and freeze-dried to obtain an organic nitroxide free radical nanocomposite.

5. The preparation method according to claim 4, characterized in that: The volume ratio of the organic solvent to the water is 1:1 to 1:10; and / or, removing the organic solvent by a solvent volatilization method, wherein the solvent volatilization method comprises: stirring at 100 to 1000 rpm / min at room temperature for 5 to 24 hours; And / or, the molar ratio of TEMP-NH2 to carboxyl groups in the nanoparticle carrier solution is 1:0.3-3.

6. The preparation method according to claim 4, characterized in that: The stirring reaction temperature of step (2) is 10-40° C., and the reaction time is 10-40 h; And / or, the molecular weight cut-off of the dialysis bag used for dialysis is 7000-20000, and the dialysis time is 12-100 hours.

7. The preparation method according to any one of claims 4 to 6, characterized in that The PLGA carrier is one or more of PLGA-COOH, PLGA-PEG, PLGA-OH, PLGA-PEG-OH, PLGA-PEG-COOH, and PLGA-PEG; and / or, TEMP-NH2 is a combination of one or more of 3-amino-2,2,6,6-tetramethylpiperidine, 4-amino-2,2,6,6-tetramethylpiperidine, and 5-amino-2,2,6,6-tetramethylpiperidine; And / or, the amidation reaction auxiliary agent is one or more of: EDC·HCl / NHS, DCC / NHS, EDC·HCl / HOBt, and DIC / HOBt.

8. Use of an organic nitroxide free radical nanocomposite according to claim 1 or prepared by the method according to claim 5 in any of the following a) to e): a) preparing a magnetic resonance imaging contrast agent; b) preparing a medicament for use in magnetic resonance imaging diagnosis of cancer; c) preparing a medicament for alleviating symptoms in cancer patients; d) preparing a drug for treating cancer; e) Quantitative detection of reactive oxygen species not for therapeutic or diagnostic purposes.

9. The use according to claim 8, characterized in that The use of the organic nitroxide free radical nanocomposite in the quantitative detection of reactive oxygen species not for the purpose of treatment or diagnosis comprises the following steps: 1) Preparation of standard curve H2O2 solutions with different concentration gradients, including an H2O2 solution with a concentration of 0, were prepared, and organic nitroxide free radical nanocomposite solutions were added thereto for incubation. T1-weighted MR scanning was then performed using a nuclear magnetic resonance imaging device. Signal values ​​were extracted from the original images to obtain a standard curve with the H2O2 solution concentration on the abscissa and the signal intensity on the ordinate, and the standard curve equation was obtained. 2) The organic nitroxide free radical nanocomplex is injected into the body, and a T1-weighted MR scan is performed using a nuclear magnetic resonance imaging device. Signal values ​​are extracted from the original image, and the reactive oxygen species concentration is determined according to the signal value intensity using a standard curve equation.

10. The use according to claim 8, characterized in that The incubation time is 1 to 6 hours; and / or, the detection limit is calculated according to the standard curve and is 20 to 50 μM.

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