Composite preparation of polypeptide C16 and angiopoietin Ang-1 and its application

Abstract

The present invention provides a composite preparation of polypeptide C16 and angiopoietin Ang-1 and its application. By taking advantage of nanomaterials and combining the anatomical and physiological characteristics of the blood-brain barrier, a new drug delivery system for the brain is developed to achieve the best effect of treating neurodegenerative diseases by penetrating the blood-brain barrier. It is a carrier that can target and transport drugs to the local inflammation. Liposomes modified with the leukocyte chemotactic peptide fMLP are a good choice for loading protein and polypeptide biological drugs, which solves the problem of drug administration of the Ang-1 + C16 composite drug.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and in particular, to a composite preparation of polypeptide C16 and angiopoietin Ang-1 and its application. Background Art

[0002] Currently, the main clinical anti-inflammatory treatment methods include adrenal corticosteroids, non-steroidal anti-inflammatory drugs, antibiotics, etc., but the curative effects are not ideal. The abuse of antibiotics, especially broad-spectrum antibiotics, can lead to dysbacteriosis in the body, causing symptoms such as vomiting and nausea, and exacerbating the condition. Antibiotics have many side effects. For example, a decrease in white blood cells will further reduce the body's disease resistance. The stimulation of antibiotics to the gastrointestinal mucosa can cause drug-induced gastritis and intestinal flora imbalance. Long-term use of antibiotics, especially high-level antibiotics, broad-spectrum or combined use of antibiotics, will kill some non-pathogenic bacteria in the intestine, resulting in flora imbalance, disrupting the microbial balance in the intestine, and causing intestinal diseases. Even pseudomembranous enteritis and opportunistic bacteria - fungal superinfection may occur. Some non-pathogenic bacteria become pathogenic due to the loss of mutual restraint.

[0003] Long-term use of glucocorticoids can cause hypercortisolism syndrome, including moon face, buffalo hump, hypertension, hirsutism, diabetes, thinning of the skin, weight gain, lower extremity edema, purple streaks, easy bleeding tendency, poor wound healing, acne, menstrual disorders, ischemic necrosis of the humeral or femoral head, osteoporosis and fractures (including spinal compression fractures and long bone pathological fractures), muscle weakness, muscle atrophy, hypokalemia syndrome, gastrointestinal irritation (nausea, vomiting), pancreatitis, peptic ulcer or perforation, inhibition of children's growth, glaucoma, cataract, benign intracranial hypertension syndrome, impaired glucose tolerance and aggravation of diabetes. Patients may have mental symptoms such as euphoria, excitement, delirium, restlessness, disorientation, or may also show inhibition. Complications mainly include infections caused by fungi, tuberculosis bacteria, staphylococcus, proteus, pseudomonas aeruginosa and various herpes viruses. After stopping the drug, patients may experience dizziness, fainting tendency, abdominal pain or back pain, low fever, loss of appetite, nausea, vomiting, muscle or joint pain, headache, fatigue, and weakness.

[0004] Long-term use of immunosuppressive therapy can cause adverse reactions in systems and organs. For example, azathioprine, mycophenolate mofetil, and mitoxantrone can lead to upper gastrointestinal bleeding, aseptic osteonecrosis, leukopenia, and liver and kidney function impairment in patients. Plasma exchange and immunomodulators have a certain effect on alleviating the recurrence of the disease, but they are expensive, and long-term application increases the economic burden on patients. In recent years, domestic and foreign scholars have also conducted a large number of exploratory studies in aspects such as traditional Chinese medicine treatment, gene therapy, and autologous hematopoietic stem cell transplantation. However, the components of traditional Chinese medicine are complex, and it is difficult to control the toxic and side effects; gene therapy itself is a double-edged sword, and the use of viral vectors is more likely to produce serious side effects. T cell vaccines and stem cell transplantation are currently still in the experimental exploration stage, with very few clinical treatment applications, and the long-term effects remain to be further observed. Therefore, there is currently a lack of an economic, safe, and long-acting treatment method.

[0005] Regardless of the mechanism that causes inflammation, it must go through the process of increased vascular permeability, adhesion of inflammatory cells to the vessel wall, penetration through the vascular endothelial cell layer, and migration from the blood vessel into the tissue. Therefore, as long as the adhesion, migration, and penetration of inflammatory cells are blocked, and the permeability of the blood-brain barrier is reduced as much as possible, inflammation can be alleviated to a considerable extent and the internal microenvironment can be improved.

[0006] The blood-brain barrier separates the central nervous system from the peripheral blood circulation and can restrict the entry of immune cells into the central nervous system. Under normal physiological conditions, very few lymphocytes infiltrate into the brain parenchyma through the blood-brain barrier. In recent years, there have been reports suggesting that even a damaged blood-brain barrier can still effectively control the recruitment of inflammatory cells into the central nervous system. Therefore, the simple physical structure damage of the blood-brain barrier cannot explain the infiltration of immune cells into the central nervous system. In addition to the physical blood-brain barrier, the infiltration of leukocytes into the central nervous system is regulated by cell adhesion molecules, chemokines, and matrix metalloproteinases. Therefore, the research on targeting the blood-brain barrier and adhesion molecules (integrins) has very important practical significance and clinical application value for the inflammatory response of the nervous system.

[0007] Integrin is not only an adhesion molecule that plays a role in maintaining mechanical stability in the interaction between cells and their surrounding environment, but also an important mediator component for the adhesion, adherence, deformation, and penetration of inflammatory cells in blood vessels through the vessel wall. Research has shown that the surfaces of various inflammatory cells such as monocytes, lymphocytes, and macrophages express multiple integrins. If these inflammatory cells need to migrate directionally through the vascular endothelial cell layer and from the blood vessel into the inflammatory tissue, it depends on the binding of the integrin molecules on the surfaces of these cells to the integrin receptors on the surfaces of vascular endothelial cells, thereby inducing rearrangement of the cytoskeleton within the inflammatory cells and deforming the cells, so that the inflammatory cells can pass through the gaps between vascular endothelial cells. If this binding is blocked, the escape and infiltration of inflammatory cells will also be prevented.

[0008] C16 (KAFDITYVRLKF) is a polypeptide on the r1 chain of laminin, an important matrix molecule on the basement membrane to which vascular endothelial cells adhere. It has the function of recognizing and specifically binding to integrin αvβ3 and α5β1. Research has found that in an in vitro culture system supplemented with C16 polypeptide, the number of T lymphocytes that cross from one side to the other side of the vascular endothelial cell layer under the chemotactic effect of IL-16 in the lower chamber of the Transwell culture plate is significantly reduced compared with the control group. The inhibitory effect of C16 on the migration of cultured THP-1 monocytes across the vascular endothelial cell layer is similar to that of directly using an αvβ3 antibody. These results indicate that C16 can not only bind to the integrin molecule αvβ3 on the surface of inflammatory cells, competitively preventing it from recognizing and binding to the integrin receptor on the surface of vascular endothelial cells, but may also bind to αvβ3 expressed on the surface of vascular endothelial cells, similarly competitively preventing it from binding to the corresponding ligand on inflammatory cells, thereby causing deformation of the inflammatory cell cytoskeleton structure and the dynamic process of migrating through the gaps between vascular endothelial cells.

[0009] Previous studies have shown that after angiopoietin-1 (Angiopoietin1, Ang-1) binds to the receptor Tie2, the Tie2 receptor is phosphorylated and functionally activated. Activated Tie2 can not only attract perivascular cells such as vascular smooth muscle cells and pericytes to surround and support endothelial cells to form a complete vascular wall, promote vascular maturation and maintain the stability of the blood-brain barrier, but also maintain the vascular structure through cell-cell tight junctions, cell-matrix interactions, etc. The regulation of vascular permeability by the Ang-1 / Tie-2 pathway is mediated by the PI3K / Akt signaling pathway in endothelial cells. Research has shown that the PI3K / Akt signaling pathway in endothelial cells has multiple regulatory effects, including anti-endothelial cell apoptosis, promoting endothelial cell migration, promoting neovascular remodeling, and inhibiting the expression of inflammatory genes. After enhancing the activity of the Ang-1 / Tie-2 signaling pathway, the intracellular PI3K / Akt phosphorylation level increases accordingly, indicating that the Ang-1 / Tie-2 pathway has an activating effect on the PI3K / Akt signaling pathway. Research has found that Ang-1 has the effects of promoting the survival of vascular endothelial cells, reducing the vascular permeability at the injury site, and inhibiting the escape of T lymphocytes, and can produce a certain synergistic effect when used in combination with C16 polypeptide, further promoting the functional recovery after injury.

[0010] It should be noted that natalizumab (an antibody and antagonist of integrin α4) has no therapeutic effect on neuromyelitis optica, while C16 polypeptide can exert its effect in animal models of neuromyelitis optica and multiple sclerosis. Presumably, the reason is not only related to different αβ binding chains, but also indicates that the effect of single immune adhesion inhibition is limited. C16 can not only competitively prevent inflammatory cells from migrating and escaping into the extravascular tissue, but also is a specific activator of integrin αvβ3 and α5β1. Activated integrin molecules can activate the PI3K / Akt signaling pathway. The activation of αvβ3 and α5β1 can also promote the phosphorylation of Tie-2 and facilitate the binding of Ang-1 / Tie-2. Conversely, Ang-1 / Tie-2 can also activate integrin through the PI3K pathway to form a positive feedback. The interaction between Tie-2 / αvβ3 and Tie-2 / α5β1 can also selectively stimulate the PI3K / Akt signaling pathway to enhance the effect of Ang-1. All these suggest that there is a mechanism of mutual activation between integrin and Ang-1 / Tie-2 in the body.

[0011] Therefore, combining the two in a certain proportion to make a mixed preparation can promote each other, enhance the effect, and complement each other. Patent WO2009114539 discloses a composite preparation of Ang-1 and C16, which regulates vascular permeability and reduces inflammatory cell infiltration. In animal experiments, complete blood count after drug administration showed that except for a slight decrease in platelet count, C16 had no inhibitory effect on the body's overall immune system. These phenomena all suggest that C16 polypeptide has the effect of inhibiting the extravasation and tissue infiltration of T lymphocytes. It can reduce local inflammatory cell infiltration but has no side effect of systemic immunosuppression, and is a relatively safe drug. Since the main action targets of C16 and Ang-1 are in blood vessels and act on vascular endothelial cells, the drug does not need to cross the blood-brain barrier into nerve tissues, which is more rapid, effective, and safe; and in brain regions with severely damaged blood-brain barriers, the C16+Ang-1 mixed preparation entering the nervous system is more likely to activate the aforementioned PI3K / AKt signaling pathway to reduce the activation of microglia, promote the survival and stability of vascular endothelial cells, and protect the tight junctions of vascular endothelial cells. The specific formulation of this drug: C16 polypeptide (dissolved in 0.3% acetic acid) solution is mixed with Ang-1 protein (dissolved in double-distilled water). Their final concentrations are both 2mg / ml. This drug has been found to be effective in a series of central nervous system inflammatory diseases such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and Parkinson's disease (PD). It is a non-specific anti-inflammatory drug that is not targeted at a specific disease and can have a therapeutic effect in neurodegenerative diseases with nervous system inflammation.

[0012] Although existing studies have shown that the Ang-1+C16 composite drug has no risk of inhibiting the overall immune system while having anti-inflammatory effects, other possible toxic side effects remain to be elucidated. At the same time, C16 polypeptide is dissolved in a slightly acidic solvent, while angiopoietin Ang-1 is dissolved in a neutral solvent. The solution is still slightly acidic after mixing, and the pH value of the medicament needs to be readjusted to about pH 7.4 suitable for the human body after filtration sterilization, but precipitation is likely to occur at this time, affecting the utilization rate of the drug. In addition, the protein mixed preparation will be destroyed when taken orally, is not easy to enter the blood vessels by intramuscular injection, and intravenous injection may cause phlebitis due to the certain vascular irritation of the slightly acidic drug solvent during long-term medication. These drawbacks greatly limit its treatment by intravascular administration.

[0013] In recent years, the development of drug delivery systems, especially nano-drug delivery systems, has shown unique advantages in enhancing efficacy, reducing toxicity, and improving patient tolerance. They are easy to degrade in vivo, non-toxic, and non-immunogenic, and have attracted increasing attention and been widely used in neurodegenerative diseases. For example, various nano-carriers such as solid lipid nanoparticles, liposomes, nanoemulsions, and micelles have been applied to the treatment of neurodegenerative diseases. As a natural endogenous nano-carrier for drug delivery, exosomes avoid clearance by the mononuclear phagocyte system and immune rejection, and are a very promising new carrier for drugs treating Alzheimer's disease. Gold nanoparticles are negatively charged and can penetrate the blood-brain barrier through endocytosis mediated by nerve growth factor receptors, which can reduce the apoptosis of dopaminergic neurons in the substantia nigra striatum and have good therapeutic effects on Parkinson's disease models in vitro and in vivo. Solid lipid nanoparticle and poly lactic-co-glycolic acid have advantages such as good biocompatibility, biodegradability, sustained release, and targeting effects, and have been approved by the US FDA for the treatment of Huntington's disease. In this system, solid lipid nanoparticles are currently recognized as excellent carriers for drug delivery, and their most outstanding advantages are reflected in high specific surface area (which can provide a higher drug loading), charged ionic surface (which helps drug binding or functionalization), and the ability to bind to ligands (such as antibodies and targeting peptides) to achieve targeted specificity. Nowadays, the research on this drug has been applied to various severely related neurodegenerative diseases such as ADS, PD, and MS. However, the drug loading of solid lipid nanoparticles is generally only 1%-5%. Although there are also reports on increasing the drug loading, too high a drug loading may lead to gelation of solid lipid nanoparticles. In addition, solid lipid nanoparticles have poor stability during storage and may exhibit phenomena such as particle size growth or drug degradation. The above problems limit the widespread application of nanoparticles (NPs). Currently, as carrier materials for drug delivery systems, biodegradable polymer materials are generally preferred. Represented by poly lactic-co-glycolic acid (PLGA), it has good biocompatibility, does not accumulate in important organs, and the intermediate product lactic acid during its metabolism in the human body is a product of in vivo sugar metabolism, and the final products are carbon dioxide and water. Therefore, it has been approved by the US FDA as a pharmaceutical excipient and is widely used in pharmaceuticals, medical engineering materials, and drug sustained release agents. As a carrier for nano-drug delivery systems, PLGA nanoparticles generally refer to particles with a diameter between 101-103 nm. Its drug delivery system entraps drugs or bioactive substances inside the nanoparticles through dissolution, encapsulation, etc., or localizes them on the surface of the nanoparticles through attachment, electrostatic adsorption, etc. The main advantages of this system are that it can reduce the number of drug administrations while ensuring the drug efficacy, thereby reducing the toxic and side effects of the drug on the body; it can achieve targeted transportation and targeted drug delivery; it can control the sustained release of drugs and extend their half-life in vivo, etc. Summary of the Invention

[0014] The object of the present invention is to provide a composite preparation of polypeptide C16 and angiopoietin Ang-1 and its application, so as to solve the technical problems in the combined administration of polypeptide C16 and angiopoietin Ang-1.

[0015] To achieve the above object, the present invention provides a composite preparation of polypeptide C16 and angiopoietin Ang-1, the active ingredient of which is a polypeptide C16-loaded liposome conjugated with fMLP and an angiopoietin Ang-1-loaded liposome conjugated with fMLP, and the molar ratio of the two is 8:1.

[0016] Preferably, the amino acid sequences of angiopoietin Ang-1 and polypeptide C16 are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively;

[0017] Angiopoietin Ang-1:

[0018] KLENYIVENMKSEMAQIQQNAVQNHTATMLEIGTSLLSQTAEQTRKLTDVETQVLNQTSRLE IQLLENSLSTYKLEKQLLQQTNEILKI, as shown in SEQ ID NO.1;

[0019] Polypeptide C16:

[0020] KAFDITYVRLKF, as shown in SEQ ID NO.2.

[0021] Preferably, the polypeptide C16-loaded liposome conjugated with fMLP or the angiopoietin Ang-1-loaded liposome conjugated with fMLP is obtained by the following preparation method: first, DSPE-PEG2000-fMLP is obtained by cross-linking reaction with DSPE-mPEG2000-NH2 (distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amino cross-linker) and fMLP as raw materials, and then it is reacted with polypeptide C16 or angiopoietin Ang-1 to obtain the product.

[0022] More preferably, the preparation method of DSPE-PEG2000-fMLP is as follows: first, DSPE-mPEG2000-NH2, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and fMLP are dissolved in methanol, and stirred at 37°C for 2 hours, and then dried to obtain the product.

[0023] Even more preferably, the dosage ratio of DSPE-mPEG2000-NH2, EDC, NHS, fMLP, and methanol is 150 mg: 10.3 mg: 6.2 mg: 25 mg: 2 mL.

[0024] More preferably, the reaction method of DSPE-PEG2000-fMLP with polypeptide C16 or angiopoietin Ang-1 is as follows: First, dissolve DSPE-mPEG2000 and DSPE-PEG2000-fMLP in a 5% glucose solution by mass concentration, then add polypeptide C16 or angiopoietin Ang-1, perform ultrasonic treatment in an ice-water bath for 15 minutes, and filter and purify with a 0.22 μm filter membrane.

[0025] Even more preferably, when reacting with angiopoietin Ang-1, the molar ratio of DSPE-mPEG2000, DSPE-PEG2000-fMLP, and the polypeptide is 6:2:1, and the mass-to-volume ratio of DSPE-mPEG2000 to the glucose solution is 39.4 mg:3 mL.

[0026] Even more preferably, when reacting with polypeptide C16, the molar ratio of DSPE-mPEG2000, DSPE-PEG2000-fMLP, and the polypeptide is 3:1:1, and the mass-to-volume ratio of DSPE-mPEG2000 to the glucose solution is 268.8 mg:22 mL.

[0027] The present invention provides the use of the above-mentioned composite preparation of polypeptide C16 and angiopoietin Ang-1 in the preparation of a therapeutic drug for central nervous system inflammatory diseases.

[0028] (The dosage of the raw materials, except that the amount of DSPE-mPEG2000-NH2 is a fixed value of 150 mg, the other components can fluctuate up and down by 1 mg, but the liposome encapsulation rate prepared according to the above ratio is the highest)

[0029] The present invention has the following beneficial effects:

[0030] fMLP is the earliest discovered leukocyte chemotactic peptide. The latest research shows that fMLP has strong leukocyte chemotactic activity. It can efficiently activate phagocytes by binding to the formyl peptide receptor (FPR) on the surface of target cells, inducing chemotactic reactions, cell degranulation, and desensitization reactions in the body, and plays an important role in the body's inflammatory response and innate immunity. Based on the characteristic of increased leukocytes during inflammation, fMLP is covalently modified on cholesterol to construct fMLP-modified liposomes, which can target and deliver polypeptide drugs to deep inflammatory sites, providing a new strategy for enhancing the delivery to inflammatory lesions. The liposomes modified by the leukocyte chemotactic peptide fMLP in this application can improve the delivery ability of liposomes to inflammatory lesions.

[0031] The present invention utilizes the advantages of nanomaterials and combines the anatomical and physiological characteristics of the blood-brain barrier to develop a new drug delivery system for the brain, achieving the best effect of penetrating the blood-brain barrier to treat neurodegenerative diseases. It is a carrier that can target the delivery of drugs to the local inflammation. The liposome modified with the leukocyte chemotactic peptide fMLP is a good choice for loading protein and polypeptide biopharmaceuticals, which solves the problem of administering the composite drug of angiopoietin Ang-1 and C16 polypeptide.

[0032] In addition to the purposes, features, and advantages described above, the present invention has other purposes, features, and advantages. The following will refer to the drawings for a further detailed description of the present invention. Brief Description of the Drawings

[0033] The drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0034] Figure 1 is the hydrodynamic size of the 12-peptide-loaded liposome conjugated with fMLP (after conjugation);

[0035] Figure 2 is the Zeta potential of the 12-peptide-loaded liposome conjugated with fMLP (after conjugation);

[0036] Figure 3 is the electron micrograph of the angiopoietin-loaded liposome conjugated with fMLP;

[0037] Figure 4 is the electron micrograph of the 12-peptide-loaded liposome conjugated with fMLP;

[0038] Figure 5 is the fluorescence spectrum of the angiopoietin-loaded liposome conjugated with fMLP;

[0039] Figure 6 is the fluorescence spectrum of the 12-peptide-loaded liposome conjugated with fMLP;

[0040] Figure 7 are the results of rat experiments, where A is the behavioral result, B is the cortical somatosensory potential, and C is the cortical motor potential;

[0041] Figure 8 are the diagrams of the C16+Ang-1 group, the 16-peptide-loaded liposome+angiopoietin-loaded liposome group, and the 16-peptide-loaded liposome conjugated with fMLP+angiopoietin-loaded liposome conjugated with fMLP group passing through the blood-brain barrier and the blood-spinal cord barrier. Among them, A-C are the diffusion of the drug through the blood-brain barrier in nerve tissue, D-F are the diffusion of the drug through the blood-spinal cord barrier in nerve tissue, G-I are the local magnifications of A-C, and J-L are the local magnifications of D-F.

[0042] Figure 9 For the experimental results on the marginal ear of New Zealand white rabbits, among which, A - B is the direct injection group of C + A, and C - D is the liposome drug - loaded group. Specific implementation manners

[0043] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but the present invention can be implemented in many different ways defined and covered by the claims.

[0044] The experimental reagents and instruments used in the present invention are shown in Table 1 and Table 2.

[0045] Table 1. Experimental reagents

[0046]

[0047] Table 2. Experimental instruments

[0048]

[0049]

[0050] Application Example 1:

[0051] Precisely weigh 150 mg of DSPE - PEG2000 - NH2, 10.3 mg of EDC, 6.2 mg of NHS, and 25 mg of fMLP, dissolve them in 2 mL of methanol, react at 37 °C for 2 h, and dry to obtain DSPE - PEG2000 - fMLP powder.

[0052] Application Example 2:

[0053] Weigh 13.1 mg of DSPE - mPEG2000 and dissolve it in 2 mL of 5% glucose solution, add 6.0 mg of angiogenin (molar ratio of DSPE - mPEG2000: angiogenin = 8:1) and a trace amount of 2 M NaOH to assist dissolution, perform water - bath ultrasonic treatment, and filter and purify through a 0.22 - μm filter membrane to obtain angiogenin - loaded liposomes.

[0054] Application Example 3:

[0055] Precisely weigh 89.6 mg of DSPE - mPEG2000 and dissolve it in 6 mL of 5% glucose solution, add 12.0 mg of 12 - peptide (molar ratio of DSPE - mPEG2000: 12 - peptide = 4:1), perform water - bath ultrasonic treatment, and filter and purify through a 0.22 - μm filter membrane to obtain 12 - peptide - loaded liposomes.

[0056] Example 1:

[0057] Accurately weigh 39.4 mg of DSPE-mPEG-2000 and 14.9 mg of DSPE-PEG2000-fMLP, dissolve them in 3 mL of 5% glucose solution, add 24.0 mg of angiopoietin (molar ratio of DSPE-mPEG-2000:DSPE-PEG2000-fMLP:angiopoietin = 6:2:1) and a small amount of 2 M NaOH to assist dissolution, perform ultrasonic treatment in a water bath, and filter and purify with a 0.22 μm filter membrane to obtain fMLP-conjugated angiopoietin-loaded liposomes.

[0058] Example 2:

[0059] Accurately weigh 101.7 mg of DSPE-PEG2000-fMLP and 268.8 mg of DSPE-mPEG2000, dissolve them in 22 mL of 5% glucose solution, add 48.0 mg of 12-peptide (molar ratio of DSPE-mPEG-2000:DSPE-PEG2000-fMLP:12-peptide = 3:1:1), perform ultrasonic treatment in a water bath, and filter and purify with a 0.22 μm filter membrane to obtain fMLP-conjugated 12-peptide-loaded liposomes.

[0060] Detection and Characterization

[0061] 1. Detection of Polypeptide Encapsulation Efficiency

[0062] The amphiphilic phospholipids mediate the dissolution of the polypeptide and form liposomes. After ultrafiltration, the filtrate is clear, transparent, and fluorescence-free, indicating that all the polypeptide is encapsulated.

[0063] 2. Detection of Zeta Potential and Hydrodynamic Size

[0064] Detection Results:

[0065] The hydrodynamic size (after conjugation) of the 12-peptide liposomes is shown in Figure 1 , and the Zeta potential (after conjugation) is shown in Figure 2 , and the specific data are shown in Table 3.

[0066] Table 3. Detection Results of Hydrodynamic Size and Zeta Potential of 12-Peptide Liposomes at Room Temperature

[0067]

[0068] The 12-peptide itself is positively charged. After being encapsulated by the phospholipids conjugated with fMLP and forming liposomes, it shows the negative charges carried by the phospholipids and fMLP.

[0069] Note: fMLP is first modified on phospholipids and then prepared into liposomes. Therefore, it is not necessary to detect the hydrodynamic size and Zeta potential before conjugation.

[0070] 4. Transmission Electron Microscopy Detection

[0071] The electron micrograph of the angiopoietin-loaded liposome conjugated with fMLP is shown in Figure 3 , according to the electron micrograph, the particle size of the liposome is about 25 - 40 nm, with regular shape and relatively uniform size.

[0072] The electron micrograph of the 12-peptide-loaded liposome conjugated with fMLP is shown in Figure 4 , according to the electron micrograph, the particle size of the liposome is about 25 - 40 nm, with regular shape and relatively uniform size, which is roughly consistent with the DLS detection result.

[0073] 5. Fluorescence spectrum detection

[0074] The fluorescence spectrum of the angiopoietin-loaded liposome conjugated with fMLP is shown in Figure 5 , the fluorescence spectrum of the 12-peptide-loaded liposome conjugated with fMLP is shown in Figure 6 . The detection results show that the maximum absorption wavelength of the angiopoietin-loaded liposome conjugated with fMLP is 601 nm, and that of the 12-peptide-loaded liposome conjugated with fMLP is 688 nm, which deviate from the theoretical values of 565 nm and 670 nm. This may be because part of the energy is consumed by vibrational relaxation when the fluorescent group is in the excited state, or because part of the energy is lost due to the collision between phospholipid molecules, polypeptides and the fluorescent group, resulting in Stokes shift.

[0075] In summary, the fluorescence spectrum detection results indicate that the polypeptide is successfully encapsulated into the liposome; the hydrodynamic size and electron microscopy detection show that the polypeptide particles are uniform, with regular shape and good dispersibility; the Zeta potential detection shows that the particle surface is negatively charged, indicating that the polypeptide is encapsulated inside the liposome, and only the negative charges carried by phospholipids and fMLP are shown on the surface.

[0076] The encapsulation efficiency of the liposome by the method of the present invention is greater than 80%.

[0077] Animal experiment

[0078] An acute EAE model was established by sensitizing DA rats with guinea pig spinal cord homogenate as an antigen.

[0079] Take the guinea pig spinal cord homogenate (GPSCH) and complete Freund adjuvant (CFA), mix them evenly and beat them into a water-in-oil emulsion as the antigen adjuvant emulsion. After anesthetizing the guinea pigs with sodium pentobarbital (50 mg / kg, intraperitoneal injection), perfuse the heart with pre-cooled ice-cold physiological saline until the effluent from the right atrium is clear. Carefully remove the guinea pig spinal cord, pick out the meninges and blood vessels, weigh it, add an equal amount of complete Freund adjuvant, and repeatedly beat it with a syringe to make a water-in-oil emulsion. After routinely disinfecting the skin at the root of the tail of DA rats, subcutaneously inject 0.2 mL of the antigen adjuvant emulsion at a single point. Animals in the normal control group were injected with the same dose of CFA + physiological saline. On the day of immunization and all days after immunization, observe the diet and activity of the rats daily and weigh them.

[0080] The nerve function scoring criteria for rats are as follows: 0 points, no obvious abnormality; 1 point, the tail is weak and flaccid; 2 points, slow movement and mild ataxia; 3 points, weakness in the hind limbs; 4 points, paralysis of the hind limbs; 5 points, paralysis of all four limbs or death.

[0081] In Figure 7 , (A): The behavioral results showed that the degree of dysfunction in experimental animals with multiple sclerosis was divided into: grade 0: asymptomatic; grade 1, partial tail drooping; grade 2, complete tail drooping; grade 3, complete tail drooping and partial paralysis of the hind limbs; grade 4, complete paralysis of the hind limbs; grade 5, near-death state. Two weeks after modeling, the dysfunction in the control group of experimental rats was mostly between grade 4 and grade 5, while in the C16 and Ang-1 conventional injection groups and the groups administered with ordinary liposome-encapsulated drugs, it was around grade 3, and the group administered with fmlp-ordinary liposome-encapsulated drugs could be reduced to around grade 2. (B, C): The electrophysiological results showed that two weeks after modeling, the cortical somatosensory potential (B) and cortical motor potential (C) in the control group of experimental rats could both show an extended latency (indicating a decrease in nerve conduction velocity) and a reduced amplitude (indicating a decrease in the number of surviving nerve fibers), while the C16 and Ang-1 conventional injection groups, the groups administered with ordinary liposome-encapsulated drugs, and the group administered with fmlp-ordinary liposome-encapsulated drugs could all play a mitigating role, and among them, the group administered with fmlp-ordinary liposome-encapsulated drugs was the most obvious.

[0082] Figure 8 Figures showing the blood-brain barrier penetration of the C16 + Ang-1 group, the liposome-encapsulated 16-peptide + liposome-encapsulated Ang-1 group, and the fMLP-conjugated liposome-encapsulated 16-peptide + fMLP-conjugated liposome-encapsulated Ang-1 group. Among them, A-C are the blood-brain barrier, D-F are the blood-spinal cord barrier, G-I are the local magnifications of A-C, and J-L are the local magnifications of D-F. From Figure 8It can be seen that both the C16 molecules (red-labeled G-I) and Ang-1 molecules (blue-labeled J-L) encapsulated by fmlp-conventional liposomes can cross the blood-brain barrier (A-C) and blood-spinal cord barrier (D-F) and enter the nerve tissue.

[0083] In Figure 9 the ear-edge experiment of New Zealand white rabbits showed that when directly injecting the C16 + Ang-1 solution (slightly acidic) into the ear-edge vein of the white rabbits, local swelling would occur, and exudation and inflammatory cell infiltration caused by irritation would appear around the blood vessels (A-B, red arrows). However, when nanoparticles were used for encapsulation, no obvious vascular irritation symptoms occurred locally (C-D).

[0084] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A composite preparation of polypeptide C16 and angiopoietin Ang-1, characterized in that, Its active ingredients are polypeptide C16 liposomes conjugated with fMLP and angiopoietin Ang-1 liposomes conjugated with fMLP, and the molar ratio of the two is 8:1; The polypeptide C16 liposomes conjugated with fMLP or the angiopoietin Ang-1 liposomes conjugated with fMLP are obtained by the following preparation method: First, DSPE-mPEG2000-NH2 and fMLP are used as raw materials for cross-linking reaction to obtain DSPE-PEG2000-fMLP, and then it is reacted with polypeptide C16 or angiopoietin Ang-1 to obtain; The preparation method of DSPE-PEG2000-fMLP is as follows: First, DSPE-mPEG2000-NH2, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and fMLP are dissolved in methanol, and stirred at 37 °C for 2 hours, and then dried to obtain; The reaction method of DSPE-PEG2000-fMLP with polypeptide C16 or angiopoietin Ang-1 is as follows: First, DSPE-mPEG2000 and DSPE-PEG2000-fMLP are dissolved in a 5% glucose solution by mass concentration, then polypeptide C16 or angiopoietin Ang-1 is added, ultrasonically treated in an ice-water bath for 15 minutes, and filtered and purified through a 0.22 μm filter membrane; The particle size of the polypeptide C16 liposomes conjugated with fMLP is 25-40 nm, and the particle size of the angiopoietin Ang-1 liposomes conjugated with fMLP is 25-40 nm; The dosage ratio of DSPE-mPEG2000-NH2, EDC, NHS, fMLP, and methanol is 150 mg: 10.3 mg: 6.2 mg: 25 mg: 2 mL; When reacting with angiopoietin Ang-1, the molar ratio of DSPE-mPEG2000, DSPE-PEG2000-fMLP, and polypeptide is 6:2:1, and the mass-to-volume ratio of DSPE-mPEG2000 to the glucose solution is 39.4 mg: 3 mL; When reacting with polypeptide C16, the molar ratio of DSPE-mPEG2000, DSPE-PEG2000-fMLP, and polypeptide is 3:1:1, and the mass-to-volume ratio of DSPE-mPEG2000 to the glucose solution is 268.8 mg: 22 mL.

2. The composite preparation according to claim 1, characterized in that, The amino acid sequences of angiopoietin Ang-1 and polypeptide C16 are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; Angiopoietin Ang-1: KLENYIVENMKSEMAQIQQNAVQNHTATMLEIGTSLLSQTAEQTRKLTDVETQVLNQTSRLE IQLLENSLSTYKLEKQLLQQTNEILKI, as shown in SEQ ID NO.1; Polypeptide C16: KAFDITYVRLKF, as shown in SEQ ID NO.

2.

3. Use of a composite preparation of a polypeptide C16 and angiopoietin Ang-1 according to any one of claims 1 to 2 in the preparation of a therapeutic drug for central nervous system inflammatory diseases.

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