A liposome loaded with a phagosome-promoting peptide, and a preparation method and application thereof

Abstract

The present application relates to the field of medicine, and particularly relates to a liposome loaded with phagolysosomal peptide, a preparation method and application thereof, so as to provide a drug for treating pancreatitis, and meanwhile improve the bioavailability of the drug. The liposome loaded with phagolysosomal peptide comprises phagolysosomal peptide and a shell coated on the periphery of the phagolysosomal peptide, the shell comprises an inner layer and an outer layer, the shell component is distearoyl phosphatidyl ethanolamine-polyethylene glycol, and there is a selenium-selenium bond between the inner layer and the outer layer. The inventor of the present application first discloses the direct effect and mechanism of the phagolysosomal peptide on the damaged pancreas of SAP, and the prepared DSSM@TN liposome can inhibit P2X7-induced mitochondrial damage, accumulation of ROS and expression of NLRP3, improve the bioavailability of the phagolysosomal peptide, significantly enhance the efficacy of the phagolysosomal peptide, and further improve the potential of the phagolysosomal peptide in preventing and treating SAP in the clinic. The DSSM@TN liposome can be used as a drug for treating pancreatitis, and meanwhile provides a new direction for the treatment research of pancreatitis.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a liposome loaded with a phagocytic peptide, its preparation method, and its application. Background Technology

[0002] Acute pancreatitis (AP) is the most common cause of hospitalization for non-malignant gastrointestinal diseases. It has a high recurrence rate, with 20% of acute pancreatitis patients progressing to severe acute pancreatitis (SAP), resulting in a mortality rate of 30%-50%. It is a global medical problem. SAP can cause systemic inflammatory response syndrome, causing significant damage to various organs and tissues, leading to a high mortality rate.

[0003] Recent studies on acute pancreatitis have shown that biliary tract disease, alcohol poisoning, pancreatic injury, pancreatic ischemia, and pancreatic duct stenosis are important causes of acute pancreatitis. The pathogenesis and pathological process of acute pancreatitis are very complex, involving not only cellular factors, immune factors, intracellular signaling, and infection, but also pancreatic enzymes, circulatory disorders, and apoptosis. Current drug treatments for acute pancreatitis mainly involve somatostatin and its analogues, protease inhibitors, and proton pump inhibitors. While these drugs can improve the patient's condition and prevent disease progression to some extent, they also have side effects such as dizziness, nausea, diarrhea, and decreased concentration, affecting the patient's physical and mental health and weakening their actual clinical efficacy, thus failing to achieve the expected therapeutic effect. Therefore, exploring new treatment methods for acute pancreatitis is an urgent problem to be solved.

[0004] The mechanisms underlying acute pancreatitis (SAP) are not yet fully understood. The development of SAP is the result of multiple pathogenic mechanisms, and the mechanisms of action of most drugs in SAP remain unclear, requiring further research. Therefore, it is necessary to further explore the direct effects and mechanisms of new drugs on SAP-damaged pancreas and identify key molecules involved in the pathogenesis of pancreatitis in order to develop better treatments for SAP.

[0005] In addition, most drugs for treating pancreatitis have short half-lives, low bioavailability, cumbersome administration methods, and large doses, which seriously affect their clinical potential. Therefore, there is an urgent need to develop a new drug delivery system to improve drug bioavailability. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a liposome loaded with tuftsin (TN) as a drug for treating pancreatitis, while improving the bioavailability of the drug.

[0007] The basic solution provided by the present invention is: a liposome loaded with a phagocytic peptide, comprising the phagocytic peptide and a shell surrounding the phagocytic peptide, wherein the shell comprises an inner layer and an outer layer, the shell being composed of distearylphosphatidylethanolamine-polyethylene glycol, and there are selenium-selenium bonds between the inner layer and the outer layer.

[0008] The working principle and advantages of this invention are as follows:

[0009] The inventors of this application discovered that decreased serum prophagokinin levels are a sign of worsening pancreatitis, and that exogenous supplementation with prophagokinin can improve SAP by modulating splenic immunity. However, prophagokinin has an extremely short half-life, requiring multiple doses, and this complex administration method limits its clinical application prospects. Therefore, the inventors investigated the direct effects and mechanisms of prophagokinin on SAP-damaged pancreas. Purine receptor 7 (P2X7) is an innate immune receptor that recognizes specific DAMPs. The inventors found that this is an immune checkpoint targeted by SAP, and that prophagokinin can exert its immunomodulatory effect through interaction with specific receptors. Through extensive practical experience and research analysis, the inventors discovered that prophagokinin can inhibit the expression of NLRP3 inflammasomes and ROS in pancreatic acinar cells through the P2X7 signaling pathway, thereby alleviating SAP. Furthermore, mitochondrial dysfunction is closely associated with inflammation and ROS in SAP. During inflammatory diseases, the P2X7 receptor leads to mitochondrial damage in monocytes and activates NLRP3. Therefore, prophagokinin can alleviate SAP by regulating inflammation through P2X7-mediated mitochondrial function. In existing research, no applications of prophagokinin in the P2X7 signaling pathway have been reported. The inventors have for the first time discovered the direct effects and mechanisms of prophagokinin on SAP-damaged pancreas and directly applied it to the treatment of SAP. This provides a solid research foundation for subsequent research on SAP treatment and the development of new uses for prophagokinin, offering new clues for pancreatitis treatment strategies and contributing to the development of more effective therapeutic drugs.

[0010] Simultaneously, the inventors designed a stable reactive oxygen species (ROS)-responsive liposome, namely DSSM@TN liposome. This liposome possesses excellent biocompatibility and can be used to deliver prophagocytic peptides to target inflamed sites in the pancreas. The liposomes of this application can improve hydrophilicity and stability, prolonging the blood circulation time of the prophagocytic peptides in vivo, thus facilitating their reach to the target site in the pancreas. Furthermore, under inflammation and ROS stimulation, the selenium-selenium bonds of the liposomes are disrupted, enabling drug release at the target site. This provides superior targeting to ROS-damaged pancreatic sites and significantly improves the bioavailability of the prophagocytic peptides. Moreover, the addition of selenium-selenium bonds not only allows them to break under inflammation and ROS stimulation, releasing the drug, but also, after improving mitochondrial function in SAP (Syndrome Atrophic Pancreatitis), inhibits ROS production, exhibiting more ideal anti-inflammatory activity.

[0011] In summary, the inventors of this application have for the first time disclosed the direct effects and mechanisms of prophagocytic peptides on SAP-damaged pancreas. Furthermore, the prepared DSSM@TN liposomes can inhibit P2X7-induced mitochondrial damage, ROS accumulation, and NLRP3 expression, improve the bioavailability of prophagocytic peptides, significantly enhance their efficacy, and further improve their potential for the prevention and treatment of SAP in clinical practice. DSSM@TN liposomes are an ideal treatment method for improving pancreatic damage and can be used as a drug for treating pancreatitis, while also providing a new direction for research on pancreatitis treatment. (DSSM as mentioned in this application is an abbreviation for DSPE-Se-Se-MPEG2000, whose full Chinese name is distearylphosphatidylethanolamine-diselement bond-polyethylene glycol 2000, where DSPE is the abbreviation for distearylphosphatidylethanolamine, Se-Se refers to the diselement bond, and MPEG2000 is the abbreviation for polyethylene glycol 2000; DSSM@TN as mentioned in this application is an abbreviation for DSPE-Se-Se-MPEG2000-TN)

[0012] Preferably, the shell contains perfluorooctyl bromide. The addition of perfluorooctyl bromide (PFOB) can improve structural stability.

[0013] The present invention also provides a method for preparing liposomes loaded with phagocytic peptides, comprising the following steps:

[0014] Step 1: Preparation of DSSM

[0015] Distearate phosphatidylethanolamine was dissolved in chloroform, and then methoxy polyethylene glycol-diselement bond-carboxyl group, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were added. After complete dissolution, the reaction was carried out at room temperature. The reaction solution was concentrated by rotary evaporation under reduced pressure, precipitated with ice-cold diethyl ether, and dried under vacuum to obtain DSSM.

[0016] Step 2: Preparation of DSSM@TN

[0017] (1) Add chloroform solution to the DSSM prepared above, then add dipalmitoylphosphatidylcholine, DSSM, cholesterol and phagocytic peptide to dissolve, then use a rotary evaporator to evaporate until the solution evaporates to form a lipid film, then add phosphate buffer to the container to dissolve the film.

[0018] (2) Add a suspension of perfluorooctane, sonicate, then centrifuge the emulsion solution, and suspend the collected precipitate in phosphate buffer to obtain the final product DSSM@TN liposomes.

[0019] Preferably, the volume ratio of methoxy polyethylene glycol-diselement bond-carboxyl group, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine in step one is 2:2:1.

[0020] Preferably, in step two (1), the rotary evaporator is rotated at a speed of 50-150 rpm.

[0021] Preferably, in step two (1), the rotary evaporator is used for rotary evaporation at a temperature of 45-55°C. In this scheme, if the rotary evaporation temperature is too low or too high, it will be detrimental to the film-forming properties and affect the stability of the lipid film. When the rotary evaporation temperature is 45-55°C, the formed lipid film is very stable.

[0022] Preferably, the ultrasound in step two (2) is performed under ice bath conditions. In this scheme, if the ultrasound is not performed under ice bath conditions, the liposomes will not be able to form a spherical structure, which is not conducive to drug encapsulation.

[0023] Preferably, the ultrasound time in step two (2) is 5-8 minutes.

[0024] The application of any of the liposomes loaded with phagocytic peptides described in this invention in the preparation of drugs for treating diseases with inflammatory pathological characteristics.

[0025] Preferably, the disease with inflammatory pathological features is acute pancreatitis or severe acute pancreatitis. Attached Figure Description

[0026] Figure 1The preparation and characterization of the support are shown in (A) schematic diagram of DSSM synthesis; (B) NMR (1H NMR) spectra of DSSM and DM; (C) FT-IR spectra of DSSM, DM and Se-Se; (D) TN standard solution and (E) HPLC analysis of DSSM@TN sample.

[0027] Figure 2 The image shows the characterization of DSSM@TN liposomes. (A) is a representative TEM image of DSSM@TN, scale bar: 100 nm; (B) is the average particle size of DSSM@TN; (C) is the average zeta potential of DSSM@TN; (D) is the particle size and zeta potential of DSSM@TN over 7 days; (E) is the cell viability of DSSM@TN at 0h, 12h, 24h, 48h, and 72h; (F) is the drug release curve of DSSM@TN at different time points within 12h under stimulation with 1μm, 10μm, and 100μm H2O2. All data are expressed as mean ± standard error. Experiments were repeated at least three times; n = 5-8 liposomes / group.

[0028] Figure 3 To evaluate the safety of the drug in vivo and its ability to target the pancreas in vitro in mice, (A) shows representative H&E staining of liver, spleen, lung, and kidney tissues after DSSM@TN treatment, scale bar: 50 μm; (BC) in vitro fluorescence images (B) and quantitative analysis (C) of the pancreas at different time points after DSSM and DSSM@TN administration to 342 model mice. All data are expressed as mean ± standard error. Experiments were repeated at least three times; n = 5-8 mice / group. *** p < 0.001 vs. DM@TN NPs group.

[0029] Figure 4 To investigate the efficacy of DSSM@TN intervention in SAP, (A) is a schematic diagram of the experimental SAP protocol; (B) is a typical image of fresh pancreatic tissue and a representative H&E staining image of the pancreas, scale bar: 50 μm; (C) is the histopathological score of pancreatic tissue; (D) is serum lipase activity; (E) is serum amylase activity; (F) is pancreatic IL-6 level; (G) is pancreatic IL-1β level. All data are expressed as mean ± standard error. The experiment was repeated at least three times; n = 5 - 8 / group. * p<0.05 and ** p<0.01 vs. blank control group; # p<0.05 and ## p<0.01 vs. model group; & p<0.05 vs. DM@TN NPs group.

[0030] Figure 5To improve mitochondrial function and inhibit oxidative stress in experimental SAP using DSSM@TN, the following data were analyzed: (A) ATP levels in pancreatic tissue; (B) typical fluorescence images and quantitative data of TMRM in primary acinar cells (scale bar: 50 μm); (C) representative DCFH2DA fluorescence images and quantitative data of primary acinar cells (scale bar: 50 μm); (D) representative images and quantitative data of MitoSOX fluorescent labeling in primary acinar cells (scale bar: 50 μm); (EG) MDA levels (E), GSH (F), and MPO (G) activities in pancreatic tissue; (H) Western blot analysis of Bax, Bcl-2, and Nrf2; (I) representative transmission electron microscopy images of pancreatic tissue. All data are expressed as mean ± standard error. Experiments were repeated at least three times; n = 5-8 animals / group. * p<0.05, ** p<0.01 and *** p<0.001 vs. blank control group; # p<0.05 and ## p<0.01 vs. SAP model group; &p<0.05 and &&p<0.01 vs. DM@TN NPs group.

[0031] Figure 6 This study aimed to demonstrate that DSSM@TN improves SAP by inhibiting P2X7 signaling. (A) shows P2X7 protein expression in pancreatic tissue; (B) shows the quantification of P2X7 protein expression; (C) shows representative immunohistochemical images of P2X7 and NLRP3 expression and distribution in pancreatic tissue (scale bar: 50 μm); (D) shows representative HE staining of pancreatic tissue after AZD treatment (scale bar: 50 μm); (E) shows pancreatic injury score; (F) shows serum lipase level; and (G) shows amylase level. All data are expressed as mean ± standard error. Experiments were repeated at least three times; n = 5-8 animals / group. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. blank control group; # p < 0.05 and ## p < 0.01 vs. SAP model group; & p < 0.05 and ns, no difference vs. DM@TNNPs group.

[0032] Figure 7To demonstrate that DSSM@TN improves SAP-induced pancreatic mitochondrial damage by inhibiting P2X7 signaling, the following images are presented: (A) Representative image of Mitotracker fluorescent labeling in acinar cells, scale bar: 50 μm; (B) Quantitative analysis of Mitotracker fluorescence; (C) Representative image of DCFH2DA fluorescent labeling in acinar cells, scale bar: 50 μm; (D) Quantitative analysis of DCFH2DA fluorescence; (E) Western blot analysis of Bax, Bcl-2, Nrf2, and HO-1 expression in pancreatic tissue; (F) Quantitative analysis of Bax, Bcl-2, Nrf2, and HO-1 expression. All results are expressed as mean ± standard error. Experiments were repeated at least three times; n = 5-8 animals / group. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. blank control group; # p < 0.05 and ## p < 0.01 vs. SAP model group.

[0033] Figure 8 This is a schematic diagram illustrating how DSSM@TN protects SAP by inhibiting the P2X7-regulated mitochondrial damage signaling pathway.

[0034] In the above figures, "Control" represents the blank control group and "SAP" represents the model group. Detailed Implementation

[0035] The following detailed explanation illustrates the specific implementation methods:

[0036] Example: DSSM@TN, a liposome loaded with a phagocytic peptide, comprising the phagocytic peptide and a shell surrounding the phagocytic peptide, the shell comprising an inner layer and an outer layer, the shell being composed of distearylphosphatidylethanolamine-polyethylene glycol, the interior of the shell being loaded with perfluorobromooctane, and the inner and outer layers being separated by selenium-selenium bonds.

[0037] The preparation method of DSSM@TN includes the following steps:

[0038] Step 1: Preparation of DSSM

[0039] Weigh 50 mg of distearate phosphatidylethanolamine and dissolve it in 10 ml of chloroform. Then add methoxy polyethylene glycol-diselement-carboxyl (MPEG2000-Se-Se-COOH), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and 4-dimethylaminopyridine (the volume ratio of MPEG2000-Se-Se-COOH, EDC, and 4-dimethylaminopyridine is 2:2:1; in this example, the amount of MPEG2000-Se-Se-COOH used is 4 ml, the amount of EDC used is 4 ml, and the amount of 4-dimethylaminopyridine used is 2 ml). After complete dissolution, react at room temperature for 0.5-1 h (the reaction time in this example is 0.5 h). Concentrate the reaction solution by rotary evaporation under reduced pressure, precipitate with 6 ml of ice-cold diethyl ether, and dry under vacuum to obtain DSSM.

[0040] Step 2: Preparation of DSSM@TN

[0041] (1) Add 20 ml of chloroform solution to the DSSM prepared above, then add 12 mg dipalmitoylphosphatidylcholine, 4 mg DSSM, 4 mg cholesterol and 1 mg phagocytic peptide to dissolve it, and then use a rotary evaporator at 50-150 rpm (80 rpm in this example) and 45-55℃ (50℃ in this example) for 0.5-1 h (evaporate until dry) until the solution evaporates to form a lipid film, and then add 3 ml of phosphate-buffered solution (PBS) to the container to dissolve the film;

[0042] (2) Add 200 μl of perfluorooctane suspension and sonicate in an ice bath using an ultrasonic device (ice bath temperature is 2-6℃, the ice bath temperature used in this example is 4℃), power is 100W, sonication time is 5-8 min (frequency is 5 seconds on and 5 seconds off, sonication time is 6 min in this example); then centrifuge the emulsion solution 3 times at 5000 rpm, each time for 5 min, and suspend the collected precipitate in 1 ml PBS to obtain the final product DSSM@TN.

[0043] Comparative example: DM@TN (DM@TN as used in this application is an abbreviation for DSPE-MPEG2000-TN)

[0044] The difference between the comparative example and the embodiment is that in step one, MPEG2000-Se-Se-COOH is replaced with MPEG2000-COOH.

[0045] I. Experimental Instruments and Reagents

[0046] (1) Experimental apparatus

[0047] Transmission electron microscope (TEM, Hitachi-7500, Japan); Ultra laser (Nanobrook Omni, Bruker Instruments Ltd., UK); 1H nuclear magnetic resonance (NMR) spectrometer (NEO-600, Bruker Biospin, Germany); infrared spectrometer (Bio-Rad FTS-6000, USA); high-speed centrifuge (centrifuge 5804R, Eppendorf, Germany); high-performance liquid chromatography with ultraviolet (UV)-visible light detector (HPLC-20A, Shimadzu, Japan).

[0048] (2) Experimental reagents

[0049] Phagocytogenic peptides and AZD9056 were purchased from MedChemExpress (New Jersey, USA); DSPE and PEG2000 were purchased from RuixiBio (Xi'an, China); cell lysis buffer, protease inhibitors, phosphatase inhibitors, cell counting kit-8, ATP assay kit, and 2',7'-dichlorodihydrodiacetic acid fluorescein assay kit were purchased from Beyotime Biotech (Shanghai, China); malondialdehyde, glutathione peroxidase, and bone marrow peroxidase assay kits were purchased from Nanjing Jiancheng Biotechnology Institute (Nanjing, China); DiR iodide was purchased from Bioss (Beijing, China); tetramethylrhodamine methyl ester was purchased from Molecular Probes (Eugene, USA); anti-Bax antibody, Bcl-2, NLRP3, and P2X7 were purchased from Proteintech (Wuhan, China); Nrf2 and HO-1 were purchased from Bioss (Beijing, China); immunohistochemical staining kits were purchased from Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China). (China); unless otherwise stated, all other chemicals are from Sigma-Aldrich (St. Louis, Missouri, USA).

[0050] Animal experiments were conducted on the examples and comparative examples, and compared with the blank control group and the model group.

[0051] The specific operating procedures for animal experiments are as follows:

[0052] All animal experiments and methods were approved by the Animal Experiment Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University (C3D6CDAE30C9). Mice were divided into cages of 4-5, with the housing temperature maintained at 23 ± 2 ℃, relative humidity set at 40%-60%, and lighting set for a 12-hour day / night cycle. Before each purchase of mice for experiments, they were housed in the above-mentioned environment for one week as an acclimatization period, during which they were fed standard laboratory drinking water and feed. Laboratory water was autoclaved, and the water in each cage was changed 3-4 times per week to ensure the water quality was safe and appropriate and to prevent accidental stoppering. The standard mouse cages and mixed sawdust bedding used in the laboratory were also autoclaved and changed twice a week. Mice were regularly observed for changes in diet, weight, activity, and health status to prevent abnormal mice from interfering with experimental results.

[0053] Step 1: Methods for constructing a mouse model

[0054] Male Blab / C mice were randomly assigned to groups (N = 5). Mice were anesthetized with 1% sodium pentobarbital (1 μL / g) via intraperitoneal injection. The mice were then fixed to a sterile operating table, and the surgical site was disinfected with povidone-iodine. An abdominal incision was made in the midline of the abdomen. The duodenum was gently pulled out and placed on sterile gauze soaked in physiological saline, fully exposing the pancreaticobiliary duct and duodenal papilla. Arterial clamps were used to clamp and close the common bile duct below the liver tissue. A small surgical traction needle was inserted through the edge of the duodenum to facilitate tissue spreading. After penetrating the duodenum with a 1 mL syringe needle, a 24 G indwelling needle was inserted into the pancreaticobiliary duct along the duodenal papilla. 3.5% sodium taurocholate saline was slowly injected at a uniform rate of 0.1 mL / min using a microinfusion pump until the dose reached 0.1 mL / 100 g. After removing the arterial clamps and traction needle, the abdomen was closed with double layers. The blank control group mice were treated with the same method and the same dose of physiological saline, and all other treatments were the same as those in the model group.

[0055] Step Two: Drug Intervention

[0056] One hour before the SAP model was established, mice were injected with 150 μg / kg of phagocytic peptide via the tail vein eight times consecutively, once per hour. Similarly, one hour before model establishment, mice were injected via the tail vein with 150 μg / kg of either the example DSSM@TN or the comparative DM@TN, and again one hour after SAP model establishment. Mice were then anesthetized with 0.2% sodium pentobarbital or dislocated at the neck 24 hours after SAP model establishment, and blood and tissues were collected.

[0057] II. Experiments to investigate the properties of liposomes

[0058] This application selects DSPE-SE-SE-MPEG2000 (DSSM) as the carrier material. Figure 1 A demonstrates the structure of DSSM and its synthesis process. Furthermore, the structures of DSPE-MPEG2000 (DM) and DSSM were verified using 1H-NMR. Figure 1 As shown in B, the characteristic peaks of MPEG2000 and DSPE are ~3.6 ppm and ~1.3 ppm, respectively.

[0059] Furthermore, Fourier transform infrared spectroscopy (FT-IR) was used to detect the chemical structures and chemical bonds of DSSM, DM, and Se-Se. For example... Figure 1 As shown in Figure C, the formation of Se-Se bonds between DSPE and MPEG2000 is confirmed. In summary, these data demonstrate the successful fabrication of DSSM and DM, laying the foundation for the fabrication of the DSSM@TN example and the comparative DM@TN.

[0060] (a) Encapsulation efficiency and drug loading of DSSM@TN

[0061] Experiment 1

[0062] Phagocytogenic peptides and liposomes were determined using HPLC, such as Figure 1 As shown in Figure D, the wavelength of the ultraviolet detector is 227 nm. Standard curves were prepared using standard solutions of different concentrations (7.8125, 15.625, 31.25, 62.5, 125): y = 47927x + 112815 (R0) 2 = 0.9989, Y is the peak area, X is the concentration), and then the DSSM@TN of Example 1 was tested to calculate the encapsulation efficiency and drug loading of the phagocytic peptide. Figure 1 As shown in E, calculations show that the encapsulation efficiency and drug loading of the phagocytic peptide are approximately 77.63% and 5.38%, respectively.

[0063] (II) Characterization of liposomes

[0064] (1) Morphological characterization of DSSM@TN

[0065] Experiment 2

[0066] The morphology and structure of the DSSM@TN in the embodiment were observed using transmission electron microscopy. Figure 2 As shown in Figure A, transmission electron microscopy reveals that the DSSM@TN of this embodiment has a regular spherical morphology and a uniform core-shell structure, indicating a good material system.

[0067] (2) Particle size and potential analysis

[0068] Experiment 3

[0069] The magnitude and zeta potential of the comparative example DM@TN and the example DSSM@TN were determined using a Zetasizer Ultra. Figure 2 As shown in Figure B, the average sizes of DSSM, the comparative example DM@TN, and the example DSSM@TN were 207.26.04 nm, 224.17.19 nm, and 214.26.84 nm, respectively, with no statistically significant differences. Furthermore, as... Figure 2 As shown in Figure C, the average zeta potentials of DSSM, the comparative example DM@TN, and the example DSSM@TN are -27.87 1.35 mV, -12.67 1.09 mV, and -18.75 1.33 mV, respectively. This indicates that the phagocytic peptide encapsulated in DSSM has suitable size, morphology, structure, and zeta potential, laying the foundation for further research.

[0070] (3) Stability of DSSM@TN

[0071] Experiment 4

[0072] The particle size and potential changes of the DSSM@TN liposomes in the example were examined over 7 days. Figure 2 As shown in Figure D, no significant changes in the particle size and zeta potential of DSSM@TN were observed within 7 days, indicating that the DSSM@TN liposomes in this example have good stability.

[0073] (III) ROS responsiveness of DSSM@TN

[0074] Experiment 5

[0075] The ROS response activity of DSSM@TN in the example was determined after stimulation with different concentrations of H2O2. Figure 2 As shown in F, with the increase of H2O2 stimulation concentration, the release of DSSM@TN in the example increased in a concentration-dependent manner, showing good ROS response activity, which laid the foundation for the good therapeutic effect of the phagocytic peptide on SAP.

[0076] (iv) In vivo biosafety and in vitro targeting capability of DSSM@TN

[0077] Experiment 6

[0078] Given the favorable physicochemical properties of DSSM@TN, this application further investigated its biosafety in vivo and its metabolism in pancreatic tissue. Mice were sacrificed at 0, 7, 14, and 21 days after DSSM@TN administration, and histopathological staining was performed on different tissues.

[0079] like Figure 3As shown in Figure A, the DSSM@TN example did not cause damage to tissues such as the liver, spleen, lungs, and kidneys, indicating that the DSSM@TN example has good biosafety. Figure 3 As shown in B and 3C, in SAP-damaged pancreatic tissue, the fluorescence signal of the DIR-labeled DSSM@TN from the example significantly increased from 15 min to 6 h, gradually weakened at 6 h, further weakened at 9 h, and disappeared completely after 12 h. In contrast, the fluorescence intensity of the comparative DM@TN decreased sharply after 15 min and was almost non-existent after 30 min. These data indicate that due to the addition of Se-Se bonds, the DSSM@TN from the example can significantly prolong the duration of the prophagocytic peptide in vivo and increase the targeted concentration in damaged pancreatic tissue.

[0080] III. Experiments demonstrating the effectiveness of liposomes

[0081] (a) Cytotoxicity

[0082] Experiment 7

[0083] Toxicity is crucial to the application prospects of drugs; therefore, this application evaluated the cytotoxicity of liposomes on primary pancreatic acinar cells. Figure 2 As shown in E, DSSM, the comparative example DM@TN, and the example DSSM@TN do not affect cell viability.

[0084] (II) Evaluation of drug efficacy

[0085] Experiment 8

[0086] like Figure 4 As shown in Figure A, 150 μg / kg of the comparative example DM@TN or the example DSSM@TN were administered 1 hour before and 1 hour after the SAP model. Figure 4 As shown in Figure B, injection of sodium taurocholate (NAT) resulted in inflammatory cell infiltration, edema, and necrosis in severely damaged areas of the pancreatic tissue; while compared to the comparative example DM@TN, the example DSSM@TN reduced pancreatic damage and demonstrated better therapeutic efficacy. Similarly, as Figure 4 As shown in Figure C, after DSSM@TN was applied to SAP, the average damage score of the pancreas was significantly reduced.

[0087] In addition, such as Figure 4 As shown in D and 4E, compared with the comparative DM@TN treatment, the DSSM@TN in this example showed better inhibition of serum lipase and amylase than the comparative DM@TN. Figure 4As shown in F and 4G, compared with DM@TN, DSSM@TN also showed a significantly greater decrease in IL-6 and IL-1β in the pancreas. These results all indicate that, compared with the comparative DM@TN, the DSSM@TN of this example has better protective potential against SAP, exhibits more ideal anti-inflammatory activity, and has broader application prospects. This is all due to the key role of the Se-Se bond in the improvement of SAP in vivo by prophagocytic peptides.

[0088] (III) DSSM@TN reduces mitochondrial damage

[0089] Experiment 9

[0090] This application investigated the effects of the DSSM@TN example on mitochondrial damage and inflammation in SAP. Figure 5 As shown in Figure A, compared to the comparative example DM@TN, the DSSM@TN example demonstrated a better effect in increasing ATP levels. Mitochondrial function is closely related to cellular ATP and inflammation; therefore, this invention uses laser scanning confocal microscopy (LSCM) to detect mitochondrial membrane potential (MMP). Figure 5 As shown in B, compared to the comparative example DM@TN, the example DSSM@TN recovered the depleted MMP to a greater extent.

[0091] Experiment 10

[0092] Mitochondrial damage directly leads to cellular oxidative stress and participates in the pathogenesis of SAP. Therefore, this application further investigates the ROS levels in acinar cells using DCFH2DA fluorescence assay. Figure 5 As shown in Figure C, the fluorescence intensity of DCFH2DA decreased in the DSSM@TN treatment group of Example C, indicating that DSSM@TN of Example C can inhibit SAP-induced oxidative stress; similarly, as Figure 5 As shown in Figure D, compared with the comparative example DM@TN, the mitochondrial ROS in the acinar cells labeled with MitoSOX dye in Example DSSM@TN was significantly reduced. Therefore, Example DSSM@TN has a better inhibitory effect on oxidative stress than the comparative example DM@TN.

[0093] In addition, such as Figure 5 As shown in E and 5F, compared with the comparative example DM@TN, the decrease in MDA level and the increase in GSH level in the example DSSM@TN also indicate that the example DSSM@TN has a better potential to reduce pancreatic oxidative damage during SAP. Meanwhile, as Figure 5 As shown in G, the myeloperoxidase (MPO) activity of the DSSM@TN example was lower than that of the comparative DM@TN, and the DSSM@TN example also exhibited better anti-inflammatory function in pancreatic tissue. Figure 5 As shown in H, the DSSM@TN example inhibits mitochondrial apoptotic damage by reducing Bax expression and increasing Bcl-2 levels, and inhibits mitochondrial oxidative stress by upregulating Nrf2 / HO-1 signaling. Furthermore, as... Figure 5 As shown in Figure I, the DSSM@TN in this example more effectively restored the integrity of the mitochondrial membrane and improved damage to internal structures such as the mitochondrial cristae. In summary, these results all indicate that DSSM@TN can significantly enhance the efficacy of prophagocytic peptides and alleviate mitochondrial damage.

[0094] (iv) Application of phagocytic peptides in the P2X7 signaling pathway

[0095] (1) DSSM@TN inhibits P2X7 activity and improves SAP

[0096] Experiment 11

[0097] The P2X7 receptor is an important innate immune receptor that plays a crucial role in the initiation and mediation of inflammation. Figure 6 As shown in A and 6B, NAT-induced SAP is accompanied by significant activation of P2X7 activity. The protective effect of DSSM@TN against pancreatic pathological damage is mediated by the regulation of P2X7 activity, which is closely related to the inflammatory response. This application found that DSSM@TN and the comparative DM@TN can inhibit P2X7 expression in the pancreas. Meanwhile, as... Figure 6 As shown in Figure C, compared with the comparative DM@TN group, the DSSM@TN group of the example showed lower distribution and expression of NLRP3, demonstrating a further reduction in inflammation.

[0098] Experiment 12

[0099] To further elucidate the protective effect of prophagocytic peptides against pancreatic injury through the regulation of P2X7, this invention administered 12.5 mg / kg of the P2X7 antagonist AZD (AZD9056, the chemical formula of AZD9056 is C10) to SAP model mice after DSSM@TN treatment via intraperitoneal injection. 24 H 36 C l2 N2O2). For example... Figure 6 As shown in D and 6E, compared to the DSSM@TN group of Example 1, the AZD+ DSSM@TN group of Example 2 significantly reduced pancreatic pathological damage and damage score, indicating that DSSM@TN of Example 2 exerts its anti-SAP-induced pancreatic pathological damage effect by inhibiting P2X7. Furthermore, as... Figure 6 As shown in F and 6G, this application also found that the AZD+ embodiment DSSM@TN further reduced serum lipase and amylase levels after DSSM@TN treatment, which further supports the above conclusions.

[0100] (2) DSSM@TN inhibits the P2X7 signaling pathway to reduce mitochondrial dysfunction

[0101] Experiment 13

[0102] Because inhibiting P2X7 activity reduces pancreatic damage, it is possible to suppress intracellular inflammatory signaling closely associated with mitochondrial dysfunction to improve pancreatitis. This application uses Mitotracker staining to detect MMPs in primary acinar cells from mice under different treatments. Figure 7 As shown in A and 7B, after administration of DSSM@TN in the examples, particularly after AZD treatment, the SAP-induced decrease in MMP was restored. Furthermore, this invention also detected DCFH2DA fluorescence in acinar cells, representing ROS levels, to assess mitochondrial function, according to… Figure 7 As shown in A and 7B, the application of AZD enhances the effectiveness of the DSSM@TN embodiment in suppressing SAP-induced ROS generation. Furthermore, as... Figure 7 As shown in C and 7D, decreased Bax expression and increased Bcl-2 expression in the pancreas were also detected after DSSM@TN treatment during SAP, and these improvements were further enhanced after AZD treatment. Improved mitochondrial function is crucial for suppressing oxidative stress in SAP. Figure 7 As shown in E and 7F, this application found that the DSSM@TN example promotes the Nrf2 / HO-1 pathway by increasing Nrf2 and HO-1 expression, and during SAP, AZD further reduces oxidative stress by increasing Nrf2 and HO-1 expression after DSSM@TN treatment. These results indicate that impaired mitochondrial function accompanied by MMP loss and elevated ROS levels is a significant cause of SAP, and the DSSM@TN example improves mitochondrial function by blocking the P2X7 signaling pathway. Therefore, as Figure 8 As shown, the DSSM@TN treatment in this embodiment can protect SAP by restoring mitochondrial function through inhibition of the P2X7-mediated mitochondrial damage signaling pathway.

[0103] In summary, this application is the first to discover the application of prophagocytic peptides in the P2X7 signaling pathway. The DSSM@TN example can inhibit SAP-induced P2X7 expression. Experiments using antagonists in this invention show that AZD (a specific P2X7 receptor inhibitor) enhances the efficacy of DSSM@TN in treating SAP. The experimental results show that AZD treatment increases the inhibition of mitochondrial oxidative stress by DSSM@TN through promoting Nrf2 / HO-1 signaling and reduces mitochondrial apoptotic damage by upregulating BCL-2 expression, thereby alleviating mitochondrial damage, energy depletion, and pancreatic tissue inflammation. This indicates that prophagocytic peptides or DSSM@TN can alleviate the pathological manifestations of pancreatitis by inhibiting P2X7-mediated mitochondrial damage signal transduction. This not only enhances the potential of prophagocytic peptides for the prevention and treatment of SAP in future clinical applications but also provides a solid research foundation for researchers and offers new clues for treatment strategies for severe pancreatitis.

[0104] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A liposome loaded with a phagocytic peptide, characterized in that: It includes a phagocytic peptide and a shell surrounding the phagocytic peptide. The shell includes an inner layer and an outer layer. The shell is composed of distearylphosphatidylethanolamine-polyethylene glycol. There is a selenium-selenium bond between the inner layer and the outer layer. Perfluorooctane is loaded inside the shell. The method for preparing the liposomes includes the following steps: Step 1: Preparation of DSSM Distearate phosphatidylethanolamine was dissolved in chloroform, and then methoxy polyethylene glycol-diselement bond-carboxyl group, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were added. After complete dissolution, the reaction was carried out at room temperature. The reaction solution was concentrated by rotary evaporation under reduced pressure, precipitated with ice-cold diethyl ether, and dried under vacuum to obtain DSSM. Step 2: Preparation of DSSM@TN (1) Add chloroform solution to the DSSM prepared above, then add dipalmitoylphosphatidylcholine, DSSM, cholesterol and phagocytic peptide to dissolve, then use a rotary evaporator to evaporate until the solution evaporates to form a lipid film, then add phosphate buffer to the container to dissolve the film. (2) Add a suspension of perfluorooctane, sonicate, then centrifuge the emulsion solution, and suspend the collected precipitate in phosphate buffer to obtain the final product DSSM@TN liposomes.

2. The liposome loaded with the phagocytic peptide as described in claim 1, characterized in that: In step one, the volume ratio of methoxy polyethylene glycol-diselement bond-carboxyl group, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine is 2:2:

1.

3. The liposome loaded with the phagocytic peptide as described in claim 2, characterized in that: In step two (1), the rotary evaporator is rotated at a speed of 50-150 rpm.

4. The liposome loaded with the phagocytic peptide as described in claim 3, characterized in that: In step two (1), the rotary evaporator is used for rotary evaporation at a temperature of 45-55℃.

5. The liposome loaded with the phagocytic peptide as described in claim 4, characterized in that: The ultrasound in step two (2) is performed under ice bath conditions.

6. The liposome loaded with the phagocytic peptide as described in claim 5, characterized in that: The ultrasound time in step two (2) is 5-8 minutes.

7. The use of liposomes loaded with phagocytic peptides as described in any one of claims 1-6 in the preparation of medicaments for treating acute pancreatitis or severe acute pancreatitis.

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