Preparation method of melanin-like nanocapsule fluorescent probe based on lung injury detection

Abstract

The invention relates to the technical field of fluorescent contrast agents, in particular to a preparation method of a melanin-like nanocapsule fluorescent probe based on lung injury detection. The nanocapsule type fluorescent probe is a melanin-like nanocapsule type fluorescent probe with a hollow structure. According to the method for preparing the melanin-like nanocapsule type fluorescent probe, the biocompatible polydopamine nanocapsule can be simply and efficiently prepared, and polydopamine has a chemical structure similar to melanin, has good biocompatibility / biodegradability, low toxic and side effects and controllable size, is easy to mark by near-infrared fluorescent dye, and can be used for preparing the melanin-like nanocapsule type fluorescent probe. And the method can be used as an effective strategy for curative effect monitoring. Besides, based on a unique hollow structure, the self weight of the polydopamine capsule is obviously reduced, and the polydopamine capsule is relatively stable in an aqueous solution, uniform in size and good in dispersity, and has a good prospect in the field of fluorescence imaging.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent contrast agents, and more specifically, to a method for preparing a melanin-like nanocapsule fluorescent probe based on lung injury detection. Background Art

[0002] Hyperoxia-induced acute lung injury (HALI) is a condition in which the body is stimulated by continuous high-concentration oxygen, which changes the permeability of alveolar cells and pulmonary blood vessels, releases a large number of inflammatory factors and cytokines to damage endothelial cells and epithelial cells, and causes diffuse alveolar and interstitial edema. In severe cases, it can develop into acute respiratory distress syndrome with a very high mortality rate. Therefore, effective treatment and monitoring of HALI is particularly important and urgent.

[0003] Fluorescence imaging can provide excellent spatial and temporal resolution, and also has the advantages of fast acquisition time, less complex instrument operation and non-invasiveness. It has great potential for application in lung tissue imaging. At present, metal organic frameworks have many properties such as porosity, large specific surface area, regular and compact arrangement structure, and are widely used in the field of fluorescent probes. However, biosafety hinders its further research. Extracellular vesicles are nanoscale vesicles with membrane structure that are actively released by cells. They have good biocompatibility and can pass through the natural physiological barrier of lung tissue. They are considered to be ideal fluorescent probe materials. However, considering the complex extraction process, low yield and high cost, the subsequent development of this biomaterial is relatively difficult, which greatly limits its long-term application as a carrier for fluorescence imaging. Therefore, it is of great significance to construct a safe and stable fluorescent probe that can achieve targeted lung tissue.

[0004] In recent years, polydopamine has been widely studied and applied in the biomedical field due to its excellent properties such as biocompatibility, hydrophilicity, redox property, and adhesion. Traditional solid polydopamine nanoparticles have a small specific surface area, resulting in poor loading capacity, while mesoporous polydopamine has a large specific surface area, but the process of adjusting its size is complicated and the yield is low. The above characteristics are not conducive to fluorescence imaging of hyperoxic lung injury. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, one aspect of the present invention is to provide a melanin-like nanocapsule fluorescent probe based on lung injury detection, wherein the nanocapsule-type fluorescent probe is a hollow structured melanin-like nanocapsule fluorescent probe based on lung injury detection.

[0006] Preferably, the particle size of the nanocapsule-type fluorescent probe is 330-980 nm.

[0007] Another aspect of the present invention is to provide a method for preparing a melanin-like nanocapsule fluorescent probe for lung injury detection, wherein the preparation method comprises the following specific steps: S1. Prepare polystyrene microspheres by soap-free emulsion polymerization, using styrene monomer as the core in water to obtain solution I, and introduce nitrogen protection in advance, add initiator to obtain mixed solution I, place mixed solution I in a water bath, and after the reaction is completed, wash with water and centrifuge to obtain a milky white suspension of polystyrene microspheres, which is stored; S2. Polydopamine was coated on the outer layer of polystyrene by a one-step method to obtain a PDA@PS core-shell structure. Dopamine was dissolved in a Tris-HCl solution and dispersed evenly by ultrasonic oscillation. The polystyrene microsphere suspension prepared in S1 was added and stirred to obtain a mixed solution II. S3. The polystyrene core in PDA@PS is removed by template method, and after centrifugation and washing, a hollow melanin-like nanocapsule is obtained. The mixed solution II prepared in S2 is washed with water, centrifuged, and dispersed in a small amount of deionized water. Tetrahydrofuran is added for ultrasonic oscillation dispersion to remove the polystyrene core. After centrifugation and washing, a melanin-like nanocapsule is obtained for use; S4. Use ultrasound to fluorescently label the melanin-like nanocapsules prepared in S3, take the new indocyanine green solution and add it to the melanin-like nanocapsule solution prepared in S3, ultrasonically oscillate, centrifuge and wash with water to obtain the melanin-like nanocapsule fluorescent probe based on lung injury detection, and store it away from light.

[0008] Preferably, the concentration of solution I in S1 is 12.5-250 g / L, and the concentration of mixed solution I is 40 g / L.

[0009] Preferably, in S1, the reaction is carried out at 70°C for 24 hours and stored at 4°C.

[0010] Preferably, the pH of the Tris-HCl solution in S2 is 8.5, the concentration is 0.1 M, the concentration of the dopamine solution is 0.1-0.3 mg / mL; and the mass ratio of dopamine to polystyrene is (10-20):1.

[0011] Preferably, a polystyrene microsphere suspension is added to S2 and stirred at room temperature for 6-12 hours.

[0012] Preferably, the ultrasonic oscillation time of tetrahydrofuran added to S3 is 0.5-1h; and the volume ratio of deionized water to tetrahydrofuran is (5-20):1.

[0013] Preferably, the mass ratio of melanin-like nanocapsules to new indocyanine green in S4 is 10:1, the ultrasonic oscillation time is 0.5 h, and the mixture is stored at 4° C. away from light.

[0014] Preferably, in S2-S4, the ultrasonic frequency is 90 KHz, the ultrasonic power is 120 W, and the temperature is 25° C. Preferably, in S3-S4, the centrifugal speed is 5000-8000 rpm, and the time is 5 min.

[0015] The beneficial effects of the present invention are as follows: The method for preparing a melanin-like nanocapsule fluorescent probe based on lung injury detection of the present invention can simply and efficiently prepare biocompatible polydopamine nanocapsules, which have the advantages of large specific surface area and simple and controllable size. It is easy to adjust the size of the nanocapsules by adjusting the feed ratio. At the same time, the hollow structure is easily labeled by near-infrared fluorescent dyes. The prepared nanocapsule-type fluorescent probe can efficiently target damaged lung tissue, improve the lung-liver ratio, further improve the fluorescence imaging effect, and can be used as an effective strategy for efficacy monitoring. In addition, based on the unique hollow structure, the polydopamine capsule significantly reduces its own weight, is relatively stable in aqueous solution, has uniform size, and has good dispersibility, and has broad prospects in the field of fluorescence imaging.

[0016] Additional aspects and advantages of the invention will become apparent from the following description, or may be learned by practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which: Figure 1 is a transmission electron microscope image of the hollow structure melanin-like nanocapsule of Example 2; Figure 2 is the infrared absorption spectrum of the hollow structure melanin-like nanocapsules of Example 2; Figure 3 This is the ultraviolet absorption spectrum of the hollow melanin-like nanocapsules obtained in Example 2 labeled with near-infrared fluorescent dyes; Figure 4 This is a solution fluorescence image of the hollow structure melanin-like nanocapsules of Example 2 labeled with near-infrared fluorescent dye; Figure 5 This is the in vitro fluorescence imaging diagram of the hollow structure melanin-like nanocapsule type fluorescent probe obtained in Example 2 in normal and HALI mouse models; Figure 6 This is a semi-quantitative fluorescence image of the hollow structure melanin-like nanocapsule fluorescent probe obtained in Example 2 in important organs (heart, liver, spleen, lung, and kidney) of control and hyperoxia-induced lung injury mice. DETAILED DESCRIPTION

[0018] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0019] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from the description. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0020] Embodiment 1 S1: Styrene monomer was dissolved in water to obtain solution I with a concentration of 12.5 g / L, and nitrogen was introduced in advance for protection. An initiator with a concentration of 40 g / L was added to obtain a mixed solution I. The mixed solution I was placed in a water bath and reacted at 70°C for 24 hours. After washing with water and centrifugation, a milky white suspension was obtained and stored at 4°C. S2: Dopamine was dissolved in Tris-HCl solution (pH 8.5, 0.1 M) to a dopamine concentration of 0.1 mg / mL, and dispersed evenly by ultrasonic oscillation. The polystyrene microsphere suspension prepared in S1 was added and stirred at room temperature for 6 h to obtain a mixed solution II. S3: The mixed solution II prepared in S2 was washed with water, centrifuged, dispersed in a small amount of deionized water, and tetrahydrofuran was added for ultrasonic oscillation dispersion for 0.5 h to remove the polystyrene core. The volume ratio of deionized water to tetrahydrofuran was 5:1. After centrifugation and washing, nanocapsules were obtained, named PC-1; S4: Take the new indocyanine green solution and add it to the melanin-like nanocapsule solution prepared in S3, perform ultrasonic oscillation, centrifuge, and wash with water to obtain the melanin-like nanocapsule type fluorescent probe, which is then stored away from light.

[0021] Embodiment 2 S1: Styrene monomer was dissolved in water to obtain solution I with a concentration of 100 g / L, and nitrogen was introduced in advance for protection. An initiator with a concentration of 40 g / L was added to obtain a mixed solution I. The mixed solution I was placed in a water bath and reacted at 70°C for 24 hours. After washing with water and centrifugation, a milky white suspension was obtained and stored at 4°C. S2: Dopamine was dissolved in Tris-HCl solution (pH 8.5, 0.1 M) to a concentration of 0.2 mg / mL, and dispersed evenly by ultrasonic oscillation. The polystyrene microsphere suspension prepared in S1 was added and stirred at room temperature for 6 h to obtain a mixed solution II. S3: The mixed solution II prepared in S2 was washed with water, centrifuged, dispersed in a small amount of deionized water, and tetrahydrofuran was added for ultrasonic oscillation dispersion for 0.5 h to remove the polystyrene core. The volume ratio of deionized water to tetrahydrofuran was 10:1. After centrifugation and washing, nanocapsules were obtained, named PC-2; S4: Take the new indocyanine green solution and add it to the melanin-like nanocapsule solution prepared in S3, perform ultrasonic oscillation, centrifuge, and wash with water to obtain the melanin-like nanocapsule type fluorescent probe, which is then stored away from light.

[0022] Embodiment 3 S1: Styrene monomer was dissolved in water to obtain solution I with a concentration of 250 g / L, and nitrogen was introduced in advance for protection. An initiator with a concentration of 40 g / L was added to obtain a mixed solution I. The mixed solution I was placed in a water bath and reacted at 70°C for 24 hours. After washing with water and centrifugation, a milky white suspension was obtained and stored at 4°C. S2: Dopamine was dissolved in Tris-HCl solution (pH 8.5, 0.1 M) to a concentration of 0.3 mg / mL, and dispersed evenly by ultrasonic oscillation. The polystyrene microsphere suspension prepared in S1 was added and stirred at room temperature for 12 h to obtain a mixed solution II. S3: The mixed solution II prepared in S2 was washed with water, centrifuged, dispersed in a small amount of deionized water, and tetrahydrofuran was added for ultrasonic oscillation dispersion for 0.5 h to remove the polystyrene core. The volume ratio of deionized water to tetrahydrofuran was 20:1. After centrifugation and washing, nanocapsules were obtained, named PC-3; S4: Take the new indocyanine green solution and add it to the melanin-like nanocapsule solution prepared in S3, perform ultrasonic oscillation, centrifuge, and wash with water to obtain the melanin-like nanocapsule type fluorescent probe, which is then stored away from light.

[0023] In the present invention, the melanin-like nanocapsule type fluorescent probe prepared in Example 2 is injected into the rat tail vein to detect the imaging effect of the melanin-like nanocapsule type fluorescent probe in mice with hyperoxia-induced lung injury.

[0024] Mice were placed in a self-made oxygen chamber and aerated with high-purity oxygen for 72 hours to construct a hyperoxia-induced lung injury model. One hour before aeration with high-purity oxygen, mice were intravenously injected with IR820-labeled nanocapsules PC-1@IR820, PC-2@IR820, and PC-3@IR820 (2 mg / mL, 200 μL). After continuous aeration with high-purity oxygen for 48 hours, mice in each group were killed, and important organs (heart, liver, spleen, lung, and kidney) of each group were taken for in vitro fluorescence imaging.

[0025] Figure 1This is a transmission electron microscope image of the hollow structure melanin-like nanocapsules of Example 2. It can be seen that the size of the hollow structure melanin-like nanocapsules is about 680 nm, the particle size is uniform, and the dispersion is good.

[0026] Figure 2 The infrared absorption spectra of the intermediate products of the preparation process of the hollow melanin-like nanocapsules in Example 2 show that PC-2 and PDA@PS-2 have the same absorption spectrum at 1628 cm -1 and 1405 cm -1 The characteristic peaks at 3026 cm -1 and 2920 cm -1 The characteristic peak at disappeared in PC-2, further indicating that the polystyrene microspheres were completely dissolved. The above data prove the successful preparation of the melanin-like nanocapsule structure.

[0027] Figure 3 This is a UV absorption curve of the hollow melanin-like nanocapsules of Example 2 labeled with near-infrared fluorescent dyes. It can be seen that PC-2@IR820 has a characteristic absorption peak of IR820 at 820nm, indicating that the nanocapsules are successfully labeled with fluorescent dyes.

[0028] Figure 4 The fluorescent image of the solution of the hollow structure melanin-like nanocapsules in Example 2 is fluorescently labeled. It can be seen that with the increase of the concentration of IR820@PC-2 solution (6.25μg / mL, 12.25μg / mL, 25μg / mL, 50μg / mL, 100μg / mL), the near-infrared fluorescence signal gradually increases.

[0029] Figure 5 This is the in vitro fluorescence imaging of the hollow structure melanin-like nanocapsule-type fluorescent probe obtained in Example 2 in a mouse model. It can be seen that compared with the fluorescence signal of the lung tissue of the control group of mice, the fluorescence signal in the lung tissue of the mice with hyperoxia-induced lung injury was significantly enhanced.

[0030] Figure 6 This is a semi-quantitative fluorescence image of the hollow structure melanin-like nanocapsule-type fluorescent probe obtained in Example 2 in important organs (heart, liver, spleen, lung, and kidney) of mice. Compared with the fluorescence intensity value of the lung tissue of the control group of mice, the fluorescence intensity value in the lung tissue of the mice with hyperoxia-induced lung injury increased significantly.

[0031] From the above, it can be seen that the melanin-like nanocapsule-type fluorescent probe prepared by the present invention has controllable size, is easy to be labeled with near-infrared fluorescent dyes, and has a higher enrichment in damaged lung tissue compared with normal lung tissue. It is suitable for early detection, accurate diagnosis and efficacy monitoring of lung diseases, and can also be used as a surgical navigation reagent, for medical diagnosis and accurate diagnosis and treatment of lung diseases.

[0032] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may also have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A melanin-like nanocapsule fluorescent probe for lung injury detection, characterized in that: The nanocapsule-type fluorescent probe is a hollow-structured melanin-like nanocapsule fluorescent probe based on lung injury detection.

2. The melanin-like nanocapsule fluorescent probe for lung injury detection according to claim 1, characterized in that: The particle size of the nanocapsule-type fluorescent probe is 330-980 nm.

3. The method for preparing a melanin-like nanocapsule fluorescent probe for lung injury detection according to claim 1, characterized in that: The specific steps of the preparation method are as follows: S1. Styrene monomer is dissolved in water to obtain solution I, and nitrogen is introduced in advance for protection, an initiator is added to obtain a mixed solution I, and the mixed solution I is placed in a water bath. After the reaction is completed, a milky white suspension of polystyrene microspheres is obtained after washing with water and centrifugation, and stored; S2. Dopamine is dissolved in Tris-HCl solution, and dispersed evenly by ultrasonic oscillation. The polystyrene microsphere suspension prepared in S1 is added and stirred to obtain a mixed solution II. S3. The mixed solution II prepared in S2 was washed with water, centrifuged, dispersed in a small amount of deionized water, and tetrahydrofuran was added for ultrasonic dispersion to remove the polystyrene core, and then centrifuged and washed to obtain melanin-like nanocapsules for use; S4. Take the new indocyanine green solution and add it to the melanin-like nanocapsule solution prepared in S3, perform ultrasonic oscillation, centrifuge and wash with water to obtain the melanin-like nanocapsule fluorescent probe based on lung injury detection, and store it away from light.

4. The method for preparing a melanin-like nanocapsule fluorescent probe for lung injury detection according to claim 3, characterized in that: The concentration of solution I in the S1 is 12.5-250 g / L, and the concentration of the mixed solution I is 40 g / L; the reaction is carried out at 70° C. for 24 hours and the mixture is stored at 4° C.

5. The method for preparing a melanin-like nanocapsule fluorescent probe for lung injury detection according to claim 3, characterized in that: The pH of the Tris-HCl solution in S2 is 8.5, the concentration is 0.1 M, the concentration of the dopamine solution is 0.1-0.3 mg / mL; the mass ratio of dopamine to polystyrene is (10-20):

1.

6. The method for preparing a melanin-like nanocapsule fluorescent probe for lung injury detection according to claim 3, characterized in that: Add the polystyrene microsphere suspension into S2 and stir at room temperature for 6-12 hours.

7. The method for preparing a melanin-like nanocapsule fluorescent probe for lung injury detection according to claim 3, characterized in that: The ultrasonic oscillation time of adding tetrahydrofuran to S3 is 0.5-1h; the volume ratio of deionized water to tetrahydrofuran is (5-20):

1.

8. The method for preparing a melanin-like nanocapsule fluorescent probe for lung injury detection according to claim 3, characterized in that: The mass ratio of the melanin-like nanocapsules to the new indocyanine green in the S4 is 10:1, the ultrasonic oscillation time is 0.5 h, and the S4 is stored at 4° C. in the dark.

9. The method for preparing a melanin-like nanocapsule fluorescent probe for lung injury detection according to claim 3, characterized in that: The ultrasonic frequency in S2-S4 is 90KHz, the ultrasonic power is 120W, and the temperature is 25°C.

10. The method for preparing a melanin-like nanocapsule fluorescent probe for lung injury detection according to claim 3, characterized in that: In S3-S4, the centrifugal speed is 5000-8000 rpm and the time is 5 min.

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