Synthesis and application of elastin fluorescent probe for early diagnosis of diabetic complications
By preparing the neutrophil elastase-specific fluorescent probe NanoNEP, the problem of NE activity detection in vivo is solved, and early diagnosis and evaluation of diabetes complications is achieved, especially in wound healing and liver application, providing an accurate diagnostic tool.
Patent Information
- Application Number
- CN202510507889.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-22
- Publication Date
- 2025-08-08
AI Technical Summary
The prior art is difficult to effectively detect neutrophil elastase (NE) activity in vivo, which leads to difficulty in early diagnosis of diabetes complications, especially the difficulty in accurately assessing wound healing and liver damage.
NanoNEP, a neutrophil elastase-specific fluorescent probe, was developed. Through the preparation method of self-assembled nanoprobes, the semi-cyanine dye modified with pentafluoropropionic anhydride was used to bind to bovine serum albumin to form NanoNEP, which was used to detect NE activity.
Successfully visualize and quantify NE activity in diabetic wound healing, provide early diagnostic tools, and in vivo imaging verification shows changes in NE concentration, achieving the evaluation of systemic diabetic inflammation and accurate identification of healthy groups and diabetes groups.
Smart Images
Figure CN120442241A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the synthesis and application of an elastic enzyme fluorescent probe for early diagnosis of diabetic complications, and belongs to the technical field of biological detection. Background Art
[0002] Diabetes mellitus is a chronic metabolic disease associated with persistent hyperglycemia that severely affects multiple organs, particularly the liver and wound healing processes. Diabetes mellitus can lead to hepatitis, fibrosis, and, in more severe cases, cirrhosis. These liver diseases not only contribute to the progression of liver disease but also exacerbate the overall disease burden, complicating the management of diabetes. In addition to liver damage, diabetes mellitus is associated with numerous serious complications, such as impaired wound healing, which significantly impacts the patient's quality of life. These unhealable wounds are often driven by persistent inflammation and a dysfunctional immune response, further complicating the diabetic condition. Diabetic foot ulcers, in particular, present a high risk of infection and amputation due to their prolonged healing time, making them a major clinical problem in diabetes management.
[0003] A major factor contributing to diabetic complications is neutrophil elastase (NE), a protease that plays a key role in inflammation and impaired tissue repair. Despite its crucial importance, measuring NE activity in vivo has been challenging due to limitations in current assays. Therefore, developing a specific assay for neutrophil elastase is crucial for the early diagnosis of diabetic complications. Summary of the Invention
[0004] The present invention provides a method for preparing a neutrophil elastase (NE)-specific fluorescent probe, comprising:
[0005] (a) 100-300 mg of hemicyanine dye (HD-NH2) was dissolved in dichloromethane, triethylamine was added, and 2,2,3,3,3-pentafluoropropionic anhydride dissolved in dichloromethane was slowly added in an ice bath. The mixture was stirred for 20-28 hours to prepare a pentafluoropropionic anhydride-modified hemicyanine dye;
[0006] (b) self-assembling the pentafluoropropionic anhydride-modified hemicyanine dye described in step (a) with bovine serum albumin to prepare the neutrophil elastase-specific fluorescent probe.
[0007] In one embodiment, the molar ratio of the 2,2,3,3,3-pentafluoropropionic anhydride to the hemicyanine dye is 2:1.
[0008] In one embodiment, the pentafluoropropionic anhydride-modified hemicyanine dye (NEP) and bovine serum albumin are self-assembled in ethanol at room temperature for at least 2 hours.
[0009] The present invention also provides a self-assembled nanoprobe NanoNEP, which is self-assembled from pentafluoropropionic anhydride-modified hemicyanine dye (NEP) and bovine serum albumin.
[0010] In one embodiment, the mass ratio of the pentafluoropropionic anhydride-modified hemicyanine dye (NEP) to bovine serum albumin is 1:1.
[0011] The present invention also provides the use of the self-assembled nanoprobe NanoNEP in preparing a product for early diagnosis of diabetic complications.
[0012] In one embodiment, the product is applicable to detection objects including but not limited to serum, liver tissue, and skin wounds.
[0013] The present invention also provides a kit for early diagnosis of diabetic complications, which contains the self-assembled nanoprobe NanoNEP.
[0014] The present invention also provides a method for detecting NE activity using the self-assembled nanoprobe NanoNEP, which comprises reacting the self-assembled nanoprobe NanoNEP with a sample containing NE at pH 7-8 and 35-37° C. for at least 40 minutes, and detecting a fluorescence signal at 720 nm.
[0015] In one embodiment, the sample to be tested includes but is not limited to serum.
[0016] Beneficial effects:
[0017] 1. This study successfully visualized and quantified NE activity in diabetic wound healing using NanoNEP for the first time. This provides key insights into the role of NE in wound healing disorders and offers a potential tool for evaluating therapeutic interventions.
[0018] 2. The present invention evaluated the effect of NanoNEP for in vivo NE imaging in a diabetic mouse model. The results showed that the NE concentration in the liver of diabetic mice was significantly increased, while the signal changes caused by metformin treatment were relatively mild, suggesting the potential role of metformin in regulating liver NE.
[0019] 3. The prepared NanoNEP was used to monitor NE activity in the serum of diabetic mice. The researchers found elevated NE levels in diabetic mice, which were partially reduced after metformin treatment. This result demonstrates the potential of NanoNEP as a diagnostic tool for assessing systemic inflammation in diabetes.
[0020] 4. The NanoNEP prepared in the present invention can also be used for the detection of serum or tissue samples from different populations, which can achieve accurate identification and diagnosis of healthy groups and diabetic groups. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 The preparation process and characterization of NanoNEP, including (a) the structure of the elastase probe and its synthesis route; (b) the mass spectrometry characterization of NEP; and (c) the nuclear magnetic resonance hydrogen spectrum characterization of NEP.
[0022] Figure 2 Characterization of the effect of NanoNEP; including: (a) absorption spectra of NEP and NEP+NE; (b) fluorescence spectra of NEP and NEP+NE; (c) photoacoustic spectra of NEP and NEP+NE; (d) absorption spectra of NanoNEP and NanoNEP+NE; (e) fluorescence spectra of NanoNEP and NanoNEP+NE; (f) photoacoustic spectra of NanoNEP and NanoNEP+NE; (g) particle size distribution of NanoNEP; (h) to (j) optimization of luminescence conditions (time, pH, temperature) of NanoNEP and NanoNEP+NE.
[0023] Figure 3 Parameter optimization for NanoNEP detection of NE; (a) to (b) fluorescence spectra and linear relationships of NanoNEP at different NE concentrations; (c) to (e) fluorescence response signals of NanoNEP to different substances; (f) to (h) detection signals of NanoNEP for NE in the presence of interfering substances.
[0024] Figure 4 The application effect of NanoNEP in liver tissue; (a) Fluorescence imaging of liver tissue of mice in different groups; (b) Statistics of fluorescence intensity of liver of mice in different groups.
[0025] Figure 5 The application effect of NanoNEP in chronic wounds; among them, (a) fluorescence imaging of wounds in different groups of mice at different stages; (b) statistical data of fluorescence intensity of wounds in different groups of mice at different stages.
[0026] Figure 6 The application effect of NanoNEP in serum samples; (a) fluorescence imaging of serum from different groups of mice; (b) statistical data of fluorescence intensity of serum from different groups of mice.
[0027] Figure 7 The application effect of NanoNEP in human tissues; (a) schematic diagram of human tissue measurement; (b) fluorescence imaging of normal and pathological tissues; (c) statistical data of fluorescence intensity of normal and pathological tissues. DETAILED DESCRIPTION
[0028] Example 1 Synthesis, Characterization and Responsiveness Testing of NanoNEP
[0029] 100-300 mg of hemicyanine dye (HD-NH2) was dissolved in 10-50 ml of dichloromethane, and 2-3 ml of triethylamine was added. Subsequently, 2,2,3,3,3-pentafluoropropionic anhydride dissolved in dichloromethane was slowly added in an ice bath; the amount of 2,2,3,3,3-pentafluoropropionic anhydride was twice the molar amount of HD-NH2. The mixture was stirred for 24 hours and then purified to obtain a solid NE probe-NEP (pentafluoropropionic anhydride-modified hemicyanine dye). The successful synthesis of NEP was verified by hydrogen nuclear magnetic resonance (NMR) spectroscopy. Figure 1 ).
[0030] During the test, it was observed that NEP exhibited poor water solubility due to the hydrophobicity of all functional groups. In order to improve the biocompatibility of NEP, NEP was self-assembled with bovine serum albumin (BSA) to form NanoNEP. The specific steps are: 100-300 mg of NEP was dissolved in 10-20 ml of ethanol, and then 100-300 mg of bovine serum albumin (BSA) was added to the solution under mild stirring conditions, and the mixed system was self-assembled at room temperature for 2 hours. The obtained nanoprobe-NanoNEP solution was dialyzed with deionized water to remove unreacted components, and finally the dynamic light scattering technology (DLS) was used to determine the hydrated particle size of the nanoparticles. This self-assembly significantly enhanced the dispersibility of the probe in aqueous solution, and the hydrated particle size of NanoNEP was approximately 32.3 nm.
[0031] 5uM NanoNEP was added to PBS at pH 7.4, and 2U / ml NE was added to detect the responsiveness of NanoNEP to NE in the buffer solution. Similar to NEP, NanoNEP exhibits absorption in the range of 500-650nm. After reacting with NE, the absorption spectrum red-shifted, and the absorption peak shifted to about 700nm. Before the addition of NE, the fluorescence signal of NanoNEP was weak, but after the addition of NE, the fluorescence intensity increased significantly, and the fluorescence peak was located near 720nm. Similarly, the photoacoustic signal of NanoNEP was very weak before the addition of NE, but after the introduction of NE, the photoacoustic signal at 700nm was significantly enhanced. These results indicate that NanoNEP retains the response characteristics of NEP while having better solubility and dispersibility in aqueous environments, making it a strong candidate for NE detection ( Figure 2 ).
[0032] Example 2 Optimization of the test conditions for NanoNEP detection of NE activity
[0033] The test conditions for detecting NE activity using NanoNEP were optimized. The specific steps were as follows: 1-30 mg of NanoNEP was dissolved in PBS, NE of equal molar concentration was added, and the fluorescence intensity of the system under different conditions was measured.
[0034] (1) Effect of reaction time on NanoNEP performance:
[0035] The reaction system was controlled at a pH of 7.4 and maintained at 37°C. Kinetic studies showed that after adding NE to NanoNEP, the fluorescence signal increased rapidly from 0 to 40 minutes and reached equilibrium at approximately 50 minutes. This indicates that NanoNEP's response to NE is time-dependent and reaches a steady state after approximately 50 minutes.
[0036] (2) Effect of pH on NanoNEP performance:
[0037] By adjusting the pH value of PBS buffer to 4-9 and reacting at 37°C for 50 min, the results showed that NanoNEP exhibited the highest enzyme activity towards NE in the pH range of 7 to 8.
[0038] (3) Effect of temperature on NanoNEP performance
[0039] The fluorescence intensity of NanoNEP for NE detection was tested at 25, 30, 35, 37, and 40°C. The results showed that the optimal response signal was obtained at 37°C, where the fluorescence signal reached its maximum, indicating that 37°C is the optimal temperature for the interaction between NanoNEP and NE. Ultimately, the optimal reaction conditions for NanoNEP detection of NE were determined to be pH 7-8 and 37°C, which yielded the most sensitive and stable NE detection response.
[0040] Example 3 Stability Study of NanoNEP Detection of NE Activity
[0041] Based on the optimal test conditions determined in Example 2, the responsiveness of NanoNEP to NE was studied in detail at pH 7.4 and 37°C. Figure 3 As shown in Figure 2, as the NE concentration increases (0-2 U / L), the fluorescence signal near 720 nm gradually increases. The relationship between the fluorescence signal and NE enzyme activity is linear, with a correlation coefficient as high as R 2=0.98916. This indicates that NanoNEP can effectively monitor changes in NE concentration based on fluorescence intensity. To evaluate NanoNEP's selectivity for NE, its responsiveness was tested in the presence of potential interfering substances, including metal ions, reactive oxygen species (ROS), and enzymes. Specifically, 50 μM metal ions (potassium, aluminum, magnesium, copper, iron, ferrous, and sodium), 50 μM reactive oxygen species (nitric oxide, singlet oxygen, peroxynitrite, hypochlorous acid, hydroxyl radicals, hydrogen peroxide, and superoxide anion), or 2 U / L enzymes (quinone oxidoreductase 1, matrix metalloproteinases, cathepsin B, alkaline phosphatase, and carboxylesterase) were added to a PBS reaction system containing 5 μM NanoNEP and 2 U / L NE.
[0042] The results showed that NanoNEP showed significant signal enhancement only in the presence of NE. Other interfering substances (metal ions 50μM, ROS 50μM and biological enzymes 2U / L) had little or no effect on the NanoNEP signal. This indicates that NanoNEP selectively interacts with NE and demonstrates its potential for specific and sensitive detection of NE in complex biological samples. These findings confirm that NanoNEP is highly responsive to NE, with a strong linear relationship between signal intensity and NE concentration, and exhibits excellent selectivity in the presence of common interfering substances. Figure 3 From 1 to 3 hours, the fluorescence intensity of NanoNEP on NE was stable and unaffected in the presence of interfering substances such as metal ions, reactive oxygen species, or enzymes. This indicates that the fluorescence signal of the probe is not interfered with by endogenous substances in the body.
[0043] Example 4 Application of NanoNPE in the Liver
[0044] The control group consisted of healthy BALB / c mice, the experimental group consisted of diabetic mice (the modeling method was referred to "ASponge-Like Double-Layer Wound Dressing with Chitosan and DecellularizedBovine Amniotic Membrane for Promoting Diabetic Wound Healing"), and the treatment group consisted of diabetic mice + metformin (treated at a dose of 200 mg / kg / day for 7 days). After successful induction of diabetes, NanoNEP was introduced into the mice by intravenous injection at an injection concentration of 200 μM and an injection dose of 100 μL, administered once a day. Subsequently, the NE concentration in the liver was monitored by photoacoustic (PA) imaging. PA imaging is a non-invasive technique that combines the high spatial resolution of ultrasound and the molecular specificity of optical light, enabling real-time, in vivo tracking of molecular biomarkers.
[0045] like Figure 4 As shown in the figure, the photoacoustic signal of healthy mice is in the range of 100-150. Over time, the photoacoustic signal of diabetic mice increased from about 120 to about 300, and that of the treatment group increased from 120 to about 230. Before the injection of the probe, the liver signals of all three groups of mice were relatively weak, indicating that they were in a state of baseline activity. However, 30 minutes after the injection, the photoacoustic signal intensity of diabetic mice increased significantly, while only a slight enhancement was observed in the control and treatment groups. This significant difference in signal intensity indicates that there are obvious differences in the physiological states of the livers of the various groups. 60 minutes after the injection, the photoacoustic signal of the liver of the treated mice was still significantly weaker than that of the diabetic group, indicating that the response of the diabetic group was more lasting.
[0046] Taken together, these findings demonstrate that photoacoustic signals evolve over time, with the diabetic group exhibiting a significant and sustained increase in signal intensity, suggesting that diabetes leads to altered NE expression. In contrast, the treated group exhibited a more modest signal increase, which may reflect the effects of metformin on NE regulation, highlighting its potential regulatory role in liver metabolic pathways.
[0047] Example 4 Application of NanoNPE in Chronic Wounds of Diabetes
[0048] To investigate the activity of NE in wound healing, three groups of mouse wound models were established: healthy mice, diabetic mice, and metformin-treated diabetic mice. In diabetic mice, a diabetic model was induced in BALB / c mice by intraperitoneal injection of 75 mg / kg of streptozotocin (STZ) for 4 consecutive days. In metformin-treated diabetic mice, diabetic mice were treated with a dose of 200 mg / kg / day for 7 days.
[0049] Wounds were induced on the backs of mice using ophthalmic scissors by making direct, approximately 0.5-cm circular wounds. These wounds were made 7 days before the experiment (position 4, P4), 4 days before the experiment (position 3, P3), 2 days before the experiment (position 2, P2), and on the day of the experiment (position 1, P1). These time points correspond to different stages of wound healing and were used to assess the temporal dynamics of NE activity in the wound environment. After wound formation, the specific probe NanoNEP was dissolved in PBS at a final concentration of 200 μM and sprayed onto the wound area at a dose of 100 μL. The mice were then incubated at 37°C for 30 minutes to allow NanoNEP to bind to NE at the wound site. After incubation, imaging was performed to observe the distribution and activity of NE in the wound area at different time points.
[0050] like Figure 5 As shown, in healthy mice, after the wound site was incubated with NanoNEP, the fluorescence signal gradually increased from position 1 (P1) to position 3 (P3), indicating that NE activity increased in the initial stage of wound healing. However, at position 4 (P4), 7 days after wound formation, the fluorescence signal decreased compared to P3 and basically returned to the same level as P1. This shows that in healthy mice, NE activity increased in the early stage of wound healing, but decreased in the later stage (7 days after wounding), reflecting the natural resolution of inflammation and the completion of tissue repair.
[0051] In contrast, in diabetic mice, the fluorescence signal continued to increase from P1 to P4, with NE activity peaking at P4 (7 days after wounding), nearly twice the fluorescence signal intensity at P4. This sustained increase in NE activity indicates that diabetic wounds exhibit a more severe and persistent inflammatory response, highlighting the impaired wound healing characteristics under diabetic conditions. NE levels also continued to increase in the diabetic treatment group, with the P3 fluorescence signal intensity nearly twice that of the healthy group.
[0052] Example 5 Application of NanoNPE in Diabetic Serum
[0053] Modeling was performed as in Example 4. Serum was obtained from three groups of mice: healthy mice, diabetic mice, and metformin-treated diabetic mice. To obtain mouse serum, mice were euthanized and whole blood was collected and allowed to clot at room temperature for 30 minutes. Serum was separated by centrifugation at 3500 rpm for 15 minutes. Serum samples were incubated with NanoNEP at a final concentration of 200 μM for 30 minutes. Fluorescence spectroscopy: Fluorescence signals were measured to quantify NE activity. IVIS imaging: In vivo imaging was performed using IVIS technology to verify the fluorescence spectroscopy results.
[0054] like Figure 6As shown, fluorescence spectral analysis of serum samples showed that there were significant differences in the fluorescence signals of the three groups of mice. The fluorescence signals of healthy mice were generally low, indicating that the NE activity in their serum was low. In contrast, the serum of diabetic mice (DM group) showed the strongest fluorescence signal, with a fluorescence intensity of about 4 times that of the healthy group and 3 times that of the treatment group, suggesting a significant increase in NE activity. The fluorescence signal of metformin-treated diabetic mice (DM+MET group) was between that of healthy mice and diabetic mice, indicating that NE activity was partially reduced after metformin treatment. The above results show that NanoNEP can accurately distinguish healthy individuals from individuals with different stages of diabetes.
[0055] Example 6 Application of NanoNPE in human tissue
[0056] Given that NanoNEP showed excellent performance in solution, in vivo, and serum tests, its potential for clinical translation was further verified. Tissue samples from healthy individuals were collected as a control group, and diabetic foot lesion tissue samples from diabetic patients were collected as an experimental group. The tissue samples were incubated with NanoNEP at 37°C for 30 minutes and then analyzed using IVIS imaging. The results showed ( Figure 7 ), with a stronger fluorescence signal in diabetic foot skin tissue (approximately twice that of healthy tissue). The increased fluorescence in diabetic foot lesions indicates higher levels of NE activity in the affected tissue, suggesting that NanoNEP can effectively detect areas of increased neutrophil elastase activity, particularly in the context of diabetic foot ulcers. These findings support the potential of NanoNEP as a diagnostic tool for monitoring inflammatory processes and tissue damage in clinical settings.
[0057] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
Claims
1. A method for preparing a neutrophil elastase-specific fluorescent probe, characterized in that: 100-300 mg of a hemicyanine dye is dissolved in dichloromethane, triethylamine is added, and then 2,2,3,3,3-pentafluoropropionic anhydride dissolved in dichloromethane is added. The mixture is stirred for 20-28 hours to obtain a hemicyanine dye modified with pentafluoropropionic anhydride. The hemicyanine dye modified with pentafluoropropionic anhydride is then self-assembled with bovine serum albumin to obtain the fluorescent probe.
2. The method according to claim 1, characterized in that The molar ratio of the 2,2,3,3,3-pentafluoropropionic anhydride to the hemicyanine dye is (1.5-2.5):
1.
3. The method according to claim 1 or 2, characterized in that The pentafluoropropionic anhydride-modified hemicyanine dye and bovine serum albumin were self-assembled in ethanol at room temperature for at least 2 hours.
4. A self-assembled nanoprobe, characterized in that: Self-assembled from pentafluoropropionic anhydride-modified hemicyanine dye and bovine serum albumin.
5. The self-assembled nanoprobe according to claim 4, characterized in that The mass ratio of the pentafluoropropionic anhydride-modified hemicyanine dye to bovine serum albumin is (0.8-1.2):
1.
6. Use of the self-assembled nanoprobe according to claim 4 or 5 in the preparation of a product for early diagnosis of diabetic complications.
7. The use according to claim 6, characterized in that The product is applicable to detection objects including but not limited to serum, liver tissue, and skin wounds.
8. A kit for early diagnosis of diabetic complications, characterized in that: Contains the self-assembled nanoprobe according to claim 4 or 5.
9. A method for detecting neutrophil elastase activity, characterized in that: Use the self-assembled nanoprobe according to claim 4 or 5 to detect neutrophil elastase.
10. The method according to claim 9, characterized in that The self-assembled nanoprobe is reacted with a sample to be tested containing neutrophil elastase at pH 7-8 and 35-37° C. for at least 40 minutes, and a fluorescence signal at 720 nm is detected.