Application of melanin-like nanocapsule medicine

Abstract

The invention relates to the technical field of biological medicine, in particular to application of melanin-like nanocapsule medicine. The melanin-like nanocapsule medicine is applied to lung injury. The melanin-like nanocapsule medicine provided by the invention improves the defects of poor solubility and low bioavailability of TPCA-1, has good biocompatibility, and can effectively remove excessive active oxygen in damaged lung tissues, obviously inhibit immune cell infiltration, obviously down-regulate the level of proinflammatory factors, improve pulmonary edema and relieve oxidative stress; the effect of treating the hyperxia-induced acute lung injury is obvious. The melanin-like nanocapsule drug does not have obvious toxic and side effects, is small in dosage, safe and sustainable in intravenous injection, can be used for treating hyperxia lung injury, and is a safe and efficient novel treatment choice.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically to the application of a melanin-like nanocapsule drug. Background Art

[0002] Hyperbaric oxygen therapy is an effective life-saving intervention in the intensive care unit for the treatment of carbon monoxide poisoning, decompression sickness and gas embolism. However, long-term exposure to a high-oxygen environment will produce a large amount of reactive oxygen species (ROS), which will induce oxidative stress, transmit oxidative damage at the levels of DNA, protein, cell membrane, etc., activate and amplify the inflammatory response, thereby damaging the defense function of lung tissue, leading to hyperoxia-induced lung injury (HALI), and even worsening to acute respiratory distress syndrome, which is manifested as severe damage to the alveolar barrier, a large amount of tissue fluid infiltration, aggravated pulmonary edema, and the body is in a state of severe hypoxia, endangering life safety.

[0003] In response to the pathological progression of HALI, TPCA-1, a small molecule drug with excellent anti-inflammatory properties, has been shown to effectively alleviate the uncontrollable inflammatory response in acute lung injury in mice. Its widespread use around the world is restricted by some factors, including high cost and single treatment regimen. In addition, due to the natural physiological barrier in the lungs, the bioavailability of this small molecule drug is low, the targeting of damaged lungs is not strong, and the drug is not water-soluble, so its therapeutic effect cannot reach the expected level. In order to achieve the best therapeutic effect and reduce the side effects of the drug, a treatment strategy targeting lung tissue is urgently needed to alleviate the oxidative stress state.

[0004] At present, nanomedicines can not only cross the physiological barriers in lung tissue, but also have the potential to solve the shortcomings and limitations of traditional antioxidant or anti-inflammatory drugs in lung delivery, further improve drug stability and reduce side effects, and can achieve multi-strategy combined treatment, that is, a single administration can achieve multiple therapeutic effects, which has important development prospects in the field of nanomedicine research. Therefore, it is crucial to develop a nanomedicine system that is simple to prepare, low in cost, has good biosafety, and can penetrate the physiological barrier of the lung. 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 an application of a melanin-like nanocapsule drug, and the application of the melanin-like nanocapsule drug in lung injury.

[0006] Preferably, the lung injury is hyperoxia-induced lung injury.

[0007] Preferably, the melanin-like nanocapsule drug consists of vesicular polydopamine and a small molecule inhibitor TPCA-1.

[0008] Preferably, the melanin-like nanocapsule drug synergistically alleviates oxidative stress through anti-oxidation and anti-inflammation.

[0009] Preferably, the melanin-like nanocapsule pharmaceutical dosage forms include injections, preparations for transtracheal instillation, and preparations for oral administration.

[0010] Preferably, the melanin-like nanocapsule drug administration routes include intratracheal administration, injection administration, and oral administration.

[0011] Preferably, the melanin-like nanocapsule drug can reduce the level of malondialdehyde in lung tissue and increase the levels of catalase, superoxide dismutase and glutathione peroxidase.

[0012] Preferably, the melanin-like nanocapsule drug can reduce the number of inflammatory cells in bronchoalveolar lavage fluid and downgrade the level of cellular inflammatory factors.

[0013] Preferably, the cellular inflammatory factors include TNF-α, IL-6 and IL-1β.

[0014] Another aspect of the present invention is to provide a method for preparing a melanin-like nanocapsule drug, wherein the specific steps of the preparation method are as follows: S1: Polystyrene microspheres were prepared by soap-free emulsion polymerization as the core; S2: Polydopamine was coated on the outer layer of polystyrene microspheres using a one-step method to obtain a PDA@PS core-shell structure; S3: Using the template method to remove the core of the polystyrene microspheres in PDA@PS to obtain nanocapsules; S4: Melanin-like nanocapsules were prepared by loading TPCA-1 using ultrasonic method.

[0015] The beneficial effects of the present invention are as follows: The melanin-like nanocapsule drug of the present invention improves the shortcomings of poor solubility and low bioavailability of TPCA-1, has good biocompatibility, can effectively remove excess reactive oxygen in damaged lung tissue, significantly inhibit immune cell infiltration, significantly downregulate the level of proinflammatory factors, improve pulmonary edema and relieve oxidative stress, and has significant effects in treating hyperoxia-induced acute lung injury. The melanin-like nanocapsule drug of the present application has no obvious toxic and side effects, is small in dosage, safe, and can be continuously injected intravenously, can be used to treat hyperoxia lung injury, and is a safe and efficient new treatment option.

[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 nanocapsule prepared by the present invention; Figure 2 It is the ultraviolet absorption curve diagram of the melanin-like nanocapsule drug prepared by the present invention; Figure 3 It is a bar graph showing the results of determination of different free radical scavenging rates of the melanin-like nanocapsule drug prepared by the present invention; Figure 4 It is a bar graph of the evaluation results of the biocompatibility of the melanin-like nanocapsule drug prepared by the present invention to cells; Figure 5 It is a bar chart of the analysis results of the contents of CAT, GSH-Px, SOD and MDA in the lung tissue of HALI mice after intravenous injection of the melanin-like nanocapsule drug prepared by the present invention; Figure 6 This is a bar graph of the analysis results of ELISA detection of TNF-α, IL-6, IL-1β, and IL-10 content in BALF of HALI mice after intravenous injection of the melanin-like nanocapsule drug prepared by the present invention; Figure 7 It is a bar graph of the analysis results of the total cell count in BALF of HALI mice after intravenous injection of the melanin-like nanocapsule drug prepared by the present invention; Figure 8 This is a staining result diagram of the pathological samples of lung tissue of HALI mice after intravenous injection of the melanin-like nanocapsule drug prepared by the present invention. 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] The following reagents or instruments without manufacturer indication are all commercially available conventional reagent products.

[0021] Dopamine hydrochloride, Aladdin Reagent (Shanghai) Co., Ltd. Styrene, Sigma-Aldrich (Shanghai) Trading Co., Ltd. Ammonium persulfate, Sigma-Aldrich (Shanghai) Trading Co., Ltd. TPCA-1, Shanghai Yuanye Biotechnology Co., Ltd. Preparation of melanin-like nanocapsule drugs: S1: Prepare polystyrene microspheres by soap-free emulsion polymerization, using styrene monomer as the core, dissolving styrene monomer in water to obtain solution I with a concentration of 100 g / L, and introduce nitrogen protection in advance, add ammonium persulfate initiator with a concentration of 40 g / L to obtain mixed solution I, place the mixed solution I in a water bath, react at 70°C for 24 hours, wash with water, centrifuge to obtain a milky white suspension, and store at 4°C; 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 (pH 8.5, 0.1 M) to a dopamine concentration of 0.2 mg / mL. Ultrasonic oscillation was used to disperse the dopamine uniformly. 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 polystyrene core in PDA@PS was removed by template method, and hollow melanin-like nanocapsules were obtained after centrifugation and washing. 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 and dispersion for 2 hours to remove the polystyrene core. The volume ratio of deionized water to tetrahydrofuran was 10:1. Nanocapsules were obtained after centrifugation and washing, and named PC; S4: TPCA-1 was loaded by ultrasound. 5 mg / mL TPCA-1 DMSO solution was added to the melanin-like nanocapsule solution prepared in S3. The solution was ultrasonically oscillated for 0.5 h. After centrifugation and washing with water, it was stored at 4°C away from light to obtain a melanin-like nanocapsule drug named PCT.

[0022] Experiment 1: Test and characterization of PC and PCT melanin nanocapsule drugs Take 20 μL of the synthesized PC stock solution and drop it on the carbon film copper grid. Let it dry naturally at room temperature and take a transmission electron microscope image. Figure 1 As shown, the size is about 680nm, with a hollow spherical capsule structure and good dispersion. Take another 50μLTPCA- and synthesized PCT stock solution and add deionized water to dilute to 2mL, add it to the quartz cuvette, and detect the UV absorption curves respectively, as shown Figure 2As shown, after PC is loaded with TPCA-1, obvious absorption can be seen at 300nm. At the same time, UV analysis shows that the loading rate of TPCA-1 in PC is about 9.35%.

[0023] Experiment 2: Antioxidant properties of PC PC stock solution was diluted in a concentration gradient to determine its catalytic activity to different free radicals (DPPH, ABTS, ·OH), such as Figure 3 As shown in the figure, PC has a broad-spectrum scavenging ability for DPPH, ABTS and ·OH. Within a certain concentration range, the scavenging ability increases with the increase of concentration.

[0024] Experiment 3: Biocompatibility of PCT nanocapsule drugs The cell counting kit-8 (CCK-8) method was used to evaluate the biocompatibility of PCT nanocapsule drugs. Figure 4 At the same time, MLE-12 cells (mouse lung epithelial cells) and RAW264.7 cells (mouse leukemia mononuclear macrophages) were cultured at 8×10 3 and 1×10 4 The cells were seeded in a 96-well plate at a density of 100 μg / well. After the cells adhered to the wall, they were incubated overnight with fresh culture medium containing different concentrations of PCT (0-100 μg / mL). The next day, the old culture medium was discarded, and CCK-8 culture medium solution (100 μL) was added to each well and incubated for 2 h. Finally, the absorbance at 450 nm was detected using an Epoch2 microplate reader (Bio Tek, USA). Figure 4 As shown, when the concentration of the nanocapsule drug PCT was 100 μg / mL, the cell survival rate remained above 90%.

[0025] Experiment 4: Animal Experiment The therapeutic effect of PCT nanocapsule drugs was evaluated by constructing a hyperoxia-induced lung injury model. The method is as follows: 5-week-old male BALB / c mice were placed in a homemade oxygen chamber for 72 hours, and the oxygen concentration was continuously monitored and maintained at about 95%, with a flow rate of 5L / min. Water and food were provided normally during the period. To ensure the therapeutic effect, the mice were administered with normal saline, PC (1mg / mL, 200μL), TPCA-1 (1mg / kg, 200μL), and PCT (equivalent to PC concentration 1mg / mL, 200μL) three times in a row. Among them, the injection of normal saline was the blank group, and the injection of PC and TPCA-1 was the control group. The first administration was 1h before hyperoxia induction. During the treatment, the drug was administered once every 24 hours. After 72h of hyperoxia induction, the BALF and lung tissues of mice in each group were collected for further analysis. The relevant results are shown in Experiments 5-7.

[0026] Experiment 5: In vivo antioxidant evaluation of PCT nanocapsule drugs As the most important antioxidant enzymes in organisms, CAT (catalase), SOD (superoxide dismutase) and GSH-Px (glutathione peroxidase) work synergistically to maintain the body in an oxidative balance. At the same time, MDA (malondialdehyde) is a typical oxidative medium that protects the body by neutralizing the production of reactive oxygen species. The lung tissue homogenates of each group of mice were taken and the above indicators were measured to reflect the ability of hPDA@TPCA-1 nanocapsule drugs to alleviate oxidative stress in lung tissues. Figure 5 The figure shows the results of the analysis of the CAT, GSH-Px, SOD and MDA contents in the lung tissue of mice after intravenous injection of nanocapsule drugs. It can be seen that compared with the mice in the hyperoxia lung injury group, the levels of CAT, SOD and GSH-Px in the lung tissue of mice in the nanocapsule drug treatment group were significantly decreased, while the level of MDA was significantly increased.

[0027] Experiment 6: Anti-inflammatory evaluation of PCT nanocapsules in vivo Downregulating the level of proinflammatory cytokines can significantly reduce lung inflammation, so BALF (bronchoalveolar lavage fluid) was taken from each group of mice to determine representative inflammatory factors, such as Figure 6 As shown in the figure, the results of ELISA detection of TNF-α, IL-6, IL-1β, and IL-10 content in mouse BALF showed that compared with the mice in the hyperoxic lung injury group, the levels of pro-inflammatory factors such as TNF-α, IL-6, and IL-1β in the mice treated with the nanocapsule were significantly decreased, and the level of anti-inflammatory factor IL-10 was significantly increased. The total number of cells in BALF was analyzed, as shown in the figure. Figure 7 As shown, compared with mice in the hyperoxic lung injury group, the total number of cells in the BALF of mice treated with the nanocapsule was significantly reduced.

[0028] Experiment 7: Protective effect of PCT nanocapsule drugs on lungs After the treatment, the lung tissues of the mice were taken for HE staining, and the pathological samples of the lung tissues of HALI mice were stained for histopathological analysis after intravenous injection of nanocapsule drugs. Figure 8 As shown, compared with mice in the hyperoxia lung injury group, the lung tissue edema of mice treated with the nanocapsule drug was significantly alleviated.

[0029] In summary, the melanin-like nanocapsule drug prepared by the present invention has a good effect in treating hyperoxia-induced lung injury. It has a significant alleviating effect in downregulating oxidative stress products in mouse lung tissue, reducing inflammatory factors and total cell counts in mouse bronchoalveolar lavage fluid, and improving the degree of lung tissue damage in mice.

[0030] 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. An application of a melanin-like nanocapsule drug, characterized in that: Application of the melanin-like nanocapsule drug in lung injury.

2. The use of a melanin-like nanocapsule drug according to claim 1, characterized in that: The lung injury is hyperoxia-induced lung injury.

3. The use of a melanin-like nanocapsule drug according to claim 1, characterized in that: The melanin-like nanocapsule drug consists of vesicular polydopamine and a small molecule inhibitor TPCA-1.

4. The use of a melanin-like nanocapsule drug according to claim 1, characterized in that: The melanin-like nanocapsule drug synergistically alleviates oxidative stress through anti-oxidation and anti-inflammation.

5. The use of a melanin-like nanocapsule drug according to claim 1, characterized in that: The melanin-like nanocapsule pharmaceutical dosage forms include injections, preparations for transtracheal instillation and preparations for oral administration.

6. The use of a melanin-like nanocapsule drug according to claim 1, characterized in that: The administration routes of the melanin-like nanocapsule drug include intratracheal administration, injection administration, and oral administration.

7. The use of a melanin-like nanocapsule drug according to claim 1, characterized in that: The melanin-like nanocapsule drug can reduce the level of malondialdehyde in lung tissue and increase the levels of catalase, superoxide dismutase and glutathione peroxidase.

8. The use of a melanin-like nanocapsule drug according to claim 1, characterized in that: The melanin-like nanocapsule drug can reduce the number of inflammatory cells in bronchoalveolar lavage fluid and downgrade the level of cellular inflammatory factors.

9. The use of a melanin-like nanocapsule drug according to claim 8, characterized in that: The cellular inflammatory factors include TNF-α, IL-6 and IL-1β.

10. The method for preparing a melanin-like nanocapsule drug according to claim 1, characterized in that: The specific steps of the preparation method are as follows: S1: Polystyrene microspheres were prepared by soap-free emulsion polymerization as the core; S2: Polydopamine was coated on the outer layer of polystyrene microspheres using a one-step method to obtain a PDA@PS core-shell structure; S3: Using the template method to remove the core of the polystyrene microspheres in PDA@PS to obtain nanocapsules; S4: Melanin-like nanocapsules were prepared by loading TPCA-1 using ultrasonic method.

Images