Inflammation microenvironment responsive cross-linked polypeptide modified liposome

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to an inflammatory microenvironment responsive cross-linked polypeptide modified liposome. The DSPE-PEG-Pep-coated Lipo-PAP-6 provided by the invention is a colon in-situ long-acting liposome with inflammatory microenvironment response aggregation, the liposome is induced by ROS (reactive oxygen species) highly expressed at a colon focus part to crosslink sulfydryl to form a disulfide bond to generate aggregation, and meanwhile, under the action of MMP-2, the inflammatory microenvironment response polypeptide is slowly dissociated to release a medicine, so that in-situ long-acting residence is realized. The DSPE-PEG-Pep-Lipo-PAP-6 can effectively improve a plurality of inflammatory indexes of a model mouse after being administered once / 2d or once / 4d, and is not obviously different from that of a Lipo-PAP-6 liposome which is not modified with a functional carrier material once / 1d, but the plurality of indexes are superior to those of an experimental group which is administered once / 1d, which shows that the DSPE-PEG-Pep-Lipo-PAP-6 can effectively improve the problems of short in-situ residence time after enema and need of frequent administration.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a liposome modified with an inflammatory microenvironment-responsive cross-linked polypeptide. Background Art

[0002] Inflammation, as an important mechanism of the innate immune system, involves the activation of immune and non-immune cells to fight pathogens and promote the repair and recovery of damaged tissues. However, if harmful substances persist or the normal healing process is hindered, acute inflammation may not subside effectively and evolve into chronic inflammation. In the medical practice of treating inflammation, oral non-steroidal anti-inflammatory drugs have always been regarded as a classic treatment. However, the efficacy of these drugs is often restricted by serious side effects such as gastric ulcers, gastric bleeding, and nausea, which greatly limits the scope and effect of their clinical application. In order to overcome these inherent problems, researchers have recently begun to explore new active pharmaceutical ingredients, such as naturally derived anti-inflammatory compounds and biomolecules, in order to develop safer and more effective treatments for inflammation. Despite this, these new drugs are not perfect, and they also have some significant shortcomings. First, the active ingredients of natural products are often difficult to accurately determine, which makes the drug development process complicated and full of uncertainty. In addition, it is worth noting that about one-third of Crohn's disease patients do not respond significantly to anti-TNF-α antibody treatment, which further highlights the challenges and obstacles faced by current inflammation treatments.

[0003] Inflammatory microenvironment-responsive cross-linked peptides are one of the current hot topics in the field of biomedicine. This type of peptide can undergo a cross-linking reaction under a specific inflammatory microenvironment, thereby changing its structure, activity or releasing drugs, and achieving effective regulation of the inflammatory process. In recent years, researchers at home and abroad have made significant progress in this field, providing new strategies and methods for the treatment of inflammation. In China, the research on inflammatory microenvironment-responsive cross-linked peptides started early and has achieved a series of important results. Researchers precisely designed the sequence and structure of peptides to enable them to specifically respond to specific molecules or signals in the inflammatory microenvironment. For example, some research teams used pro-inflammatory enzymes as targets and designed cross-linked peptides that can respond to their activity. These peptides can specifically bind to pro-inflammatory enzymes at the site of inflammation and undergo a cross-linking reaction under their catalysis, thereby achieving effective intervention in the inflammatory process. In addition, domestic researchers have also actively explored drug delivery strategies based on inflammatory mediators, and achieved precise release and regulation of drugs by designing cross-linked peptides that can respond to changes in the levels of inflammatory mediators. These research results provide new ideas and methods for the treatment of inflammation and have important clinical application value. Internationally, research on inflammatory microenvironment-responsive cross-linked peptides is also active and shows a trend of diversification. Scientific research teams in developed countries such as Europe and the United States have a high level of research and rich experience in this field. Through advanced synthesis technology and characterization methods, they have successfully prepared a series of cross-linked peptides with excellent performance, and have deeply studied their response mechanisms and biological effects in the inflammatory microenvironment. In addition, international researchers also focus on the combined application of cross-linked peptides with other therapeutic methods, such as cross-integration with nanotechnology, genetic engineering and other fields, and have developed a series of innovative and practical biomedical applications. These research results not only enrich the understanding of cross-linked peptides responsive to the inflammatory microenvironment, but also provide valuable experience and inspiration for future research. However, despite significant progress in the research of cross-linked peptides responsive to the inflammatory microenvironment at home and abroad, there are still some challenges and problems. First, the stability and biocompatibility of peptides are key issues that need to be solved. In the inflammatory microenvironment, peptides may be affected by a variety of factors and degrade or inactivate, so it is necessary to further optimize their design and improve their stability and biocompatibility. Secondly, large-scale production and cost control are also important factors restricting their practical application. At present, the synthesis and purification process of cross-linked peptides is relatively complex, the cost is high, and it is difficult to achieve large-scale production. Therefore, it is necessary to explore simpler and more efficient synthesis methods to reduce production costs and promote its widespread clinical application.

[0004] In order to solve the problems of poor mucosal penetration, short in situ residence time and insufficient long-term strategies, a long-term in situ resident liposome was designed to achieve rapid entry into the inflammatory site and responsive aggregation. Epithelial cells, neutrophils, macrophages, etc. in the local inflammatory lesions are affected by inflammatory mediators and release reactive oxygen species (ROS) 10-100 times more than normal tissues. Nanoparticles modified with peptides with exposed thiol groups at the end can be oxidized by ROS to generate disulfide bonds (-SS-), thereby cross-linking the monodisperse nanoparticles that penetrate the mucosa and enter the interstitial tissue to form an assembly to delay the release of drugs. Since inflammatory mediators such as TNF-α and IL-6 can stimulate the overexpression of MMP-2 in the inflammatory lesions, polymer micelles modified with MMP-2-sensitive peptides (PLGVRG) can responsively break the GV peptide bond in the colitis microenvironment and control the release of drugs in the lesions. Based on the specificity of the above-mentioned inflammatory pathophysiology, it is proposed to modify the MMP-2-sensitive PLGVRG polypeptide, increase the number of amino acids in the peptide chain to improve the hydrophilicity of the polypeptide, introduce cysteine ​​at the carbon end and expose the thiol group; at the same time, the thiol cross-linking is used to increase the steric hindrance to affect the effect of MMP-2 on the enzyme cleavage site, slow down the degradation rate of the functional polypeptide, and design a functional polypeptide with rapid responsive aggregation of ROS and slow degradation of MMP-2. Liposomes modified with functional polypeptides can effectively promote penetration in the lesion site, prolong the duration of drug action, and reduce the number of drug administrations. After administration of the inflammatory microenvironment-responsive peptide long-acting liposomes, the nanoparticles are small in size and negatively charged, so they can quickly disperse into the colon mucus layer, and penetrate the epithelial cells and reside in the inflammatory tissue under the action of PEG transport and colon EPR effect. The functional peptide modified on the surface of the nanoparticles is oxidatively cross-linked by H2O2 or OH in the inflammatory tissue to form SS, and the cross-linked liposome nanoparticles and the PEG long chains present a continuous alternating affinity-disappointment structure and form aggregates, achieving the purpose of PAP-6 in situ residence. Because the GV bond sensitive to MMP-2 is close to the connecting end of the functional peptide and PEG, after the formation of the aggregates, the enzymatic cleavage activity of MMP-2 on the GV bond is slowed down by steric hindrance, so that the long-acting liposomes loaded with cockroach peptides are released slowly, which prolongs the action time of the drug and meets the long-acting requirements.

[0005] The research team has previously selected DSPE-PEG-PepB as the functional material for subsequent delivery system research. The template peptide PLGVRG that responds to MMP-2 fractures contains mostly hydrophobic amino acids, so after coupling with DSPE-PEG to form a carrier material, a triblock polymer of DSPE (hydrophobic)-PEG (hydrophilic)-Pep (hydrophobic) is formed. In the aqueous solution system, the polypeptide end with active thiol groups will be close to DSPE, reducing the cross-linking effect. The MADIL-TOF-MS mass spectrometry results after oxidation can further confirm this inference. At the same time, when modifying liposomes, the polypeptide at the hydrophobic end will also be inserted into the phospholipid layer, which will then affect the aggregation and cross-linking function of the liposomes. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a liposome modified with a cross-linked polypeptide responsive to an inflammatory microenvironment.

[0007] The technical scheme of the present invention is as follows: a liposome DSPE-PEG-Pep@Lipo-PAP-6 modified with an inflammatory microenvironment-responsive cross-linked polypeptide, comprising: a drug, soybean lecithin, cholesterol and DSPE-PEG-Pep B, wherein the drug, soybean lecithin and cholesterol form the liposome Lipo-PAP-6, DSPE-PEG-Pep B is modified on the surface of the liposome, the DSPE-PEG-Pep B is obtained by reacting DSPE-PEG-NHS and Pep B, and the amino acid sequence of Pep B is PLGVRGGGGC.

[0008] In some embodiments, the drug is selected from active ingredients having anti-colitis effects, selected from mesalazine, sulfasalazine, racecadotril, sodium para-aminosalicylate, dexamethasone, methylprednisolone, azathioprine or PAP-6.

[0009] In some embodiments, when the drug, soybean lecithin and cholesterol form liposomes, the mass ratio of the drug, soybean lecithin and cholesterol is 0.635:2.5:12.5.

[0010] In some embodiments, the mass ratio of DSPE-PEG-Pep B to Lipo-PAP-6 is 1:20.

[0011] The present invention provides a method for preparing the inflammatory microenvironment responsive cross-linked polypeptide modified liposome, comprising the following steps:

[0012] 1) Preparation of DSPE-PEG-Pep B: Weigh DSPE-PEG-NHS and Pep B, dissolve in PBS, stir at room temperature in the dark, take out the reaction solution, dialyze in distilled water and freeze-dry;

[0013] 2) Preparation of Lipo-PAP-6: weigh soybean lecithin and cholesterol, add dichloromethane to dissolve them, evaporate and remove dichloromethane to form a lipid film, weigh PAP-6, prepare a solution with PBS, pour it into the lipid film, hydrate it with constant temperature oscillation to obtain a preliminary product of Lipo-PAP-6, extrude the preliminary product through a liposome extruder with a microporous filter membrane to obtain Lipo-PAP-6;

[0014] 3) Preparation of DSPE-PEG-Pep@Lipo-PAP-6: Take Lipo-PAP-6, add DSPE-PEG-PepB in proportion, incubate for 4 hours, and prepare DSPE-PEG-Pep@Lipo-PAP-6.

[0015] Preferably, in step 1), the molar ratio of DSPE-PEG-NHS to Pep is 1.2:1.

[0016] Preferably, the hydration condition in step 2) is hydration at 30° C. on a constant temperature oscillator at 200 rpm for 0.75 h.

[0017] Preferably, the incubation condition in step 3) is 37° C. for 4 hours.

[0018] The liposome modified with the inflammatory microenvironment-responsive cross-linked polypeptide of the present invention is used in the preparation of a drug for treating colitis.

[0019] Beneficial effects of the present invention: The DSPE-PEG-Pep@Lipo-PAP-6 provided by the present invention is a colon in situ long-acting liposome with inflammatory microenvironment response aggregation. The liposome forms disulfide bonds under the induction of ROS highly expressed in the colon lesion site, and the inflammatory microenvironment response polypeptide slowly dissociates and releases the drug under the action of MMP-2, thereby achieving long-term in situ retention. According to the pharmacodynamic study of DSPE-PEG-Pep@Lipo-PAP-6, it was found that administration once / 2d or once / 4d can effectively improve multiple inflammatory indicators of model mice, and there is no significant difference compared with the Lipo-PAP-6 liposomes of unmodified functional carrier materials administered once / 1d, but multiple indicators are better than the experimental group administered once / 1d, which shows that the constructed PAP-6 colon in situ long-acting liposome can effectively improve the problem of short in situ retention time after enema and the need for frequent administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Synthesis route of functional peptide Pep

[0021] Figure 2 Synthesis route of functional material DSPE-PEG-Pep

[0022] Figure 3Purity and molecular weight identification of functional peptides

[0023] Figure 4 This is the H NMR spectrum of DSPE-PEG-Pep on Pep

[0024] Figure 5 Mass spectrum of DSPE-PEG-Pep after cross-linking

[0025] Figure 6 This is the investigation diagram of the addition amount of DSPE-PEG-Pep

[0026] Figure 7 In vitro validation of ROS-responsive aggregation

[0027] Figure 8 Cellular uptake diagram of DSPE-PEG-Pep@Lipo-PAP-6

[0028] Fig. 9 The in vitro release results of DSPE-PEG-Pep@Lipo-PAP-6

[0029] Fig.10 Characterization of DSPE-PEG-Pep@Lipo-PAP-6 residence in the colon and its colonic tissues

[0030] Fig.11 Effect of DPP on the changes of DAI scores in UC mice

[0031] Fig.12 The effect of DPP on the thymus index and spleen index of UC mice

[0032] Fig.13 The effect of DPP on colon length and CMDI in UC mice

[0033] Fig.14 The effect of DPP on cytokines in colon tissue of UC mice

[0034] Fig.15 The effect of DPP on HS and PAS in the colon of UC mice

[0035] Fig.16 The effect of DPP on tight junction proteins in the colon of UC mice

[0036] Fig.17 The effect of DPP on the NF-κB / NLRP3 / Caspase-1 pathway in UC mice DETAILED DESCRIPTION

[0037] The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.

[0038] Preparation Example 1 Synthesis, purification, identification, and in vitro characterization of responsive cross-linking of functional polypeptide Pep

[0039] Since P, L, and V amino acids are all hydrophobic amino acids, when the functional peptide is coupled with DSPE-PEG, a DSPE (hydrophobic)-PEG (hydrophilic)-Pep (hydrophobic) triblock polymer will be formed. It is difficult to modify the surface of the amphiphilic phospholipid membrane. Therefore, adding a hydrophilic amino acid C to the carbon end of the template peptide PLGVRG can improve the hydrophilicity of the functional peptide. Figure 1 , two functional peptides were designed, with the sequences being: PepA: PLGVRGC; PepB: PLGVRGGGGC.

[0040] Synthesis of functional peptides

[0041] (1) Amino acid condensation: Place the resin (2-ChlorotritylChlorideResin) in a reaction tube with a sand core, add dichloromethane (DCM, 15 mL / g), and shake for 30 min; filter out the solvent through the sand core, add a 3-fold molar excess of amino acids with side chain protecting groups, add DMF to dissolve, then add a 10-fold molar excess of N,N-diisopropylethylamine (DIEA), and shake for 60 min; seal with methanol;

[0042] (2) Deprotection: remove DMF, add 20% piperidine DMF solution for 5 min, remove and add 20% piperidine DMF solution for 15 min; remove the piperidine solution, take a few resin pellets, wash with ethanol 3 times, add ninhydrin detection reagent for detection, heat at 105°C-110°C for 5 min, and turn dark blue for positive reaction; continue to wash the resin with DMF twice, DCM twice, and DMF twice;

[0043] (3) Continue to add amino acids for condensation: add the next protected amino acid in 3-fold excess and HBTU in 3-fold excess, both dissolved in as little DMF as possible, add to the reaction tube, immediately add DIEA in 10-fold excess, react for 30 minutes; take a few resins, wash with ethanol 3 times, add detection reagent for detection, heat at 105℃-110℃ for 5 minutes, colorless for negative reaction; continue to wash the resin with DMF once, DCM twice, and DMF twice; repeat the above deprotection operation, and connect the amino acids in the sequence from right to left. After the last amino acid is synthesized, remove the Fmoc protecting group; wash the resin with DMF twice, methanol twice, DMF once, DCM twice, and drain for 10 minutes.

[0044] (4) Collecting peptides: Prepare a cutting solution in the proportion of TFA 95%, water 1%, EDT 2%, and TIS 2%. Cut for 120 min. Blow dry the lysate with nitrogen as much as possible. Precipitate and wash six times with -80°C pre-frozen ice ether. Then evaporate the ether in a vacuum drying oven.

[0045] Purification of functional peptides

[0046] The crude peptide was purified by high performance liquid chromatography (HPLC) using a Waters BEH300 C18 column ( , 10μm); acetonitrile (A)-0.1% trifluoroacetic acid water (B); 0-3min, 15%-15% A; 3-20min, 15%-95% A; 20-24min, 95%-15% A; flow rate 1mL / min; detection wavelength 220nm; injection volume 10μL; column temperature 25℃. The collected target peptide solution was pre-frozen at -80℃ for 12h, then placed in a freeze dryer for drying, and the obtained white powder was the monomeric peptide, which was stored at -20℃ for later use.

[0047] Identification of functional peptides

[0048] Identification of peptides: HPLC was used to verify the purity of the peptides, and ESI-MS mass spectrometry was used to detect the molecular weight of the peptides. Liquid phase column: Symmetrix ODS-R (4.6×250mm, 5μm); 0.1% acetonitrile (A)-0.1% trifluoroacetic acid water (B); 0-30min, 5%-30% A; flow rate 1.0mL / min; detection wavelength 220nm; injection volume 10μL; column temperature 25℃. Mass spectrometry conditions: ion source is ESI source; Probe Bias: +4.5kv; gas flow rate: 1.5L / min; Detector: 1.5kv; CDL temperature: -20.0v; CDL temperature: 250℃

[0049] Synthesis and identification of DSPE-PEG-Pep

[0050] Weigh DSPE-PEG-NHS and Pep (molar ratio 1.2:1) accurately, dissolve in PBS, and stir at room temperature for 24 hours with a magnetic stirrer in the dark. Take out the reaction solution, put it into a dialysis bag (MW3KD), dialyze in 2L distilled water for 48 hours, freeze-dry, and store at -20℃. Dissolve the sample in deuterated DMSO for 1 H-NMR structural analysis.

[0051] In vitro characterization of DSPE-PEG-Pep responsive cross-linking - detection of free thiol groups

[0052] Reagent preparation: Accurately weigh 0.1982 g of DTNB and prepare it into 50 mL (10 mM) solution with 50 mM PBS (pH = 7.4), store it in a brown bottle, keep it in a dark place at low temperature for later use, dilute it 4 times (2.5 mM) before use, and prepare it before use; weigh DSPE-PEG-Pep and prepare it into 20 mg / mL solution with 50 mM PBS (pH = 7.4); Accurately weigh L-cysteine ​​reference substance and prepare it into 0.0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1.0 mM solutions in sequence with 50 mM PBS (pH = 7.4).

[0053] Detection of free thiol groups: Take 1.0 mL of the above-mentioned reference solution of each concentration and add it to 1.0 mL of DTNB solution (2.5 mM) pre-warmed in water at 25°C, shake well, let it stand for 10 minutes, and measure the absorbance (A) at a wavelength of 412 nm. Draw a standard curve Y=0.4221X-0.0056 (R 2 =0.9977), linear range 0 μg~1.0 mM. Take the DSPE-PEG-Pep sample to be tested and use it as self-control, and calculate the content of active group thiol according to the standard curve.

[0054] UV Characterization

[0055] A 20 mg / mL DSPE-PEG-Pep solution was prepared, and hydrogen peroxide solution was added to make the final concentration 100 μM. The mixture was incubated at 37°C for 2 h. The reaction solution was taken out and placed in a dialysis bag (MW5000). After dialysis in 2 L of distilled water for 24 h, a sample was taken and characterized by UV-visible spectroscopy and compared with the initial DSPE-PEG-Pep solution to see whether the maximum absorption wavelength had red-shifted or blue-shifted.

[0056] Mass Spectrometry Characterization The dialyzed samples were freeze-dried, and an appropriate amount was dissolved in methanol-water solution for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MADIL-TOF-MS) analysis.

[0057] result

[0058] After solid phase synthesis and HPLC purification, Pep-A and Pep-B with a purity of 90% were obtained. The molecular weights of Pep-A and Pep-B were identified by mass spectrometry as 700.5 and 871.4, respectively. The results were consistent with the calculated molecular weights, confirming that the chemical structures of the two peptides were correct. The results are shown in Figure 3

[0059] According to the 1H-NMR spectrum ( Figure 4) is the peak of the DSPE-terminal fatty hydrogen atom in DSPE-PEG, and δ=3.5~4.0ppm (a, -CH2-CH2-in DSPE-PEG units) is the peak of the PEG segment hydrogen atom in DSPE-PEG; it can be clearly seen in the figures that δ=1.5ppm (d, in Pepunits) is the characteristic peak of the sulfhydryl hydrogen atom on the polypeptide, and δ=3.0~3.5ppm (b, in Pep units) is the characteristic peak of the peptide bond hydrogen atom on the polypeptide; it can be clearly seen in the figures that δ=2.0~2.2ppm (e, in Pep units) is the characteristic peak of the nitrogen terminal hydrogen atom of the polypeptide, which disappears or significantly decreases in the figure after synthesis, proving that the polymer DSPE-PEG-Pep is successfully synthesized.

[0060] Detection results and comparison of free active groups

[0061] According to the DTNB colorimetric test, the PBS solutions of DSPE-PEG-PepA and DSPE-PEG-PepB have free thiol groups, and the contents of free thiol groups in the 20 mg / mL solution are 0.40 mM and 0.35 mM, respectively.

[0062] UV characterization results: After being treated with 100 μM hydrogen peroxide for 1 hour, the maximum wavelength of UV absorption of the two functional carrier materials was significantly red-shifted, indicating that the ROS-responsive thiol groups increased the steric hindrance of the carrier materials after cross-linking to form disulfide bonds.

[0063] Mass spectrometry characterization results and comparison: MADIL-TOF-MS test found that the mass spectra of the two functional carrier materials Figure 5 Both showed obvious bell-shaped distribution peaks, which are the characteristics of repeating units in the polyethylene glycol (PEG) structure; after oxidation treatment, new response peaks appeared in the mass spectra of the two functional carrier materials, and the molecular weight was about twice that before treatment, which proved that the two functional materials can responsively cross-link under the action of hydrogen peroxide. According to the mass spectrometry response value, it can be seen that the cross-linking effect of DSPE-PEG-PepB is more obvious.

[0064] Summary of comparison results

[0065] The cross-linking function of the functional peptides after DSPE-PEG coupling was investigated by in vitro ROS responsive stimulation, and DSPE-PEG-PepB was selected as the functional material for subsequent delivery system research. The template peptide PLGVRG that responds to MMP-2 fractures has a large number of hydrophobic amino acids, so after coupling with DSPE-PEG to form a carrier material, a triblock polymer of DSPE (hydrophobic)-PEG (hydrophilic)-Pep (hydrophobic) was formed. In the aqueous solution system, the peptide end with active thiol groups will be close to DSPE, which will reduce the cross-linking effect. The MADIL-TOF-MS mass spectrometry results after oxidation can further confirm this inference. At the same time, when modifying liposomes, the peptides at the hydrophobic end will also be inserted into the phospholipid layer, which will then affect the aggregation and cross-linking function of the liposomes. When the hydrophilicity of Pep is increased, the oxidative cross-linking effect of DSPE-PEG-PepB is enhanced, and a new response peak with a higher intensity than DSPE-PEG-PepA appears in its mass spectrometry results, indicating that the liposomes modified by DSPE-PEG-PepB will have better aggregation function in inflammatory sites with high ROS expression.

[0066] Preparation Example 2 Preparation of Lipo-PAP-6

[0067] Preparation of Lipo-PAP-6: Weigh soybean lecithin and cholesterol accurately, place them in a 100mL eggplant-shaped bottle, add 5mL of dichloromethane to fully dissolve them, remove dichloromethane by rotary evaporation at 200rpm on a rotary evaporator, and form a uniform lipid film on the bottle wall. Weigh 3.0mg of PAP-6, prepare a 1.0mg / mL solution with PBS (pH=7.4), pour it into the above-mentioned eggplant-shaped bottle, oscillate and hydrate at 200rpm at 30℃ constant temperature oscillator for 0.75h to obtain the initial product of Lipo-PAP-6. Extrude the initial product of Lipo-PAP-6 twice through a liposome extruder with a 0.20μm microporous filter membrane to obtain a milky Lipo-PAP-6 suspension, seal it at 4℃, and store it away from light. PAP-6 is a polypeptide extracted from cockroaches. Previous studies have shown that it has a significant anti-colitis effect. Its sequence is LAEASQAADESERAR.

[0068] Preparation process research

[0069] When preparing Lipo-PAP-6, it was found that the drug-lipid ratio had a great influence on the encapsulation efficiency of cockroach polypeptides. Therefore, under the optimal process conditions, the effects of drug-lipid ratios of 1:7.5, 1:10, 1:15, 1:20, and 1:30 on particle size, potential, and encapsulation efficiency were investigated, and the optimal prescription process conditions were selected to prepare Lipo-PAP-6.

[0070] Free drug removal and encapsulation efficiency determination method

[0071] PAP-6 is a monomer drug. It is more direct and accurate to measure the encapsulation rate by directly detecting the content of PAP-6. After preparing Lipo-PAP-6, methanol was added to make the organic phase ratio 30%, 50%, and 70%, respectively. Ultrasonic demulsification was performed for 15 minutes, and the content of PAP-6 was tested by HPLC after passing through a 0.22μm filter membrane. Calculate the polypeptide recovery rate of Lipo-PAP-6 (in terms of polypeptide recovery rate). Lipo-PAP-6 polypeptide recovery rate (%) = Lipo-PAP-6 polypeptide amount / total input × 100%

[0072] Accurately pipette 1.0mL Lipo-PAP-6 into a 100KD dialysis bag, dialyze at 200rpm in 2L PBS solution (pH 6.5), take samples from the bag at 0, 2, 4, 6, 8, 10, 12h, and 24h to detect the PAP-6 drug content after demulsification, end the dialysis at the time point when the PAP-6 content is stable, and calculate the PAP-6 encapsulation rate. PAP-6 encapsulation rate (%) = detected amount at the end of dialysis / detected amount at the beginning of dialysis × 100%

[0073] result

[0074] The experiment found that the proportion of methanol had the greatest impact on the recovery rate of peptides. When the proportion of methanol was 30%, 50%, and 70%, the recovery rates of PAP-6 were 90.0%, 63.9%, and 19.4%, respectively. Therefore, a methanol proportion of 30% and ultrasound for 15 minutes were selected as the conditions for liposome demulsification. Since the hydrophilic drugs inside and outside the phospholipid membrane in the liposome suspension system are often in a state of dynamic equilibrium, the dialysis time can be shortened by selecting a large-pore dialysis membrane and increasing the volume of the dialysis fluid. According to the determination of the content, particle size, and volume of the dialyzed liposome solution at different time points, the content of PAP-6 gradually decreased with the extension of time at first, and there was no obvious change when the dialysis time was 4h to 6h, and there was a rapid decrease after more than 6h; the particle size of Lipo-PAP-6 did not change significantly within 12h, and the volume of the liposome solution did not change significantly within 24h. Therefore, dialysis for 4h was selected as a method to remove free drugs.

[0075] The particle sizes of Lipo-PAP-6 prepared with different drug-lipid ratios are significantly different. When the drug-lipid ratio is greater than 1:15, the particle size is basically stable at about 237nm, the PDI is less than 0.300, and the surface potential is less than -11.0mV, indicating that the liposomes are relatively uniform and stable; and as the PAP-6 drug ratio increases, the particle size of Lipo-PAP-6 increases significantly, and the surface potential decreases significantly. At the same time, as the drug ratio increases, the PAP-6 encapsulation rate shows a trend of first increasing and then slightly decreasing. When the drug-lipid ratio is 1:15, the encapsulation rate is the highest, which is 63.5%, so the drug-lipid ratio of Lipo-PAP-6 is selected as 1:15.

[0076] The suspension dilutions of Lipo-PAP-6 before dialysis and after 4 hours of dialysis were taken and tested with a Zeta particle size potential meter. The results showed that the average particle size before dialysis was 237±6.3nm, PDI0.293±0.04, and Zeta potential was -11.1±0.3mV; the average particle size after dialysis was 222.6±3.4nm, PDI0.197±0.02, and Zeta potential was -15.0±0.1mV. Before and after dialysis, the particle size and morphology of the liposomes did not change significantly, and they were all spherical or quasi-spherical with internal depressions.

[0077] Example Preparation of DSPE-PEG-Pep@Lipo-PAP-6

[0078] Take the above-mentioned Lipo-PAP-6 with free drugs removed, add DSPE-PEG-Pep at a ratio of 0.5, 1, 2.5, 5, and 10%, incubate at 37°C for 4 hours, and prepare DSPE-PEG-Pep@Lipo-PAP-6, and test the effect of the amount of DSPE-PEG-Pep added on its particle size and potential. Take another liposome modified with different ratios of DSPE-PEG-Pep, and incubate it in a hydrogen peroxide solution with a final concentration of 100 μM for 2 hours. According to the change in particle size, the addition of DSPE-PEG-Pep is further preferred.

[0079] Characterization of DSPE-PEG-Pep@Lipo-PAP-6

[0080] Particle size, potential

[0081] Nanoparticle size potentiometry and transmission electron microscopy were used to characterize the characteristics of DSPE-PEG-Pep@Lipo-PAP-6.

[0082] ROS-responsive aggregation

[0083] Hydrogen peroxide solution with a final concentration of 100 μM was added to DSPE-PEG-Pep@Lipo-PAP-6 and incubated at 37°C. Samples were taken at 0, 1, 2, 4, 8, 12, and 24 h, and the particle size was measured using a nanoparticle size potentiometer. Samples with significant changes in particle size were selected for sample preparation and observation under transmission electron microscopy.

[0084] In vitro uptake experiments

[0085] PAP-6 (FITC-PAP-6) peptide was labeled with FITC to prepare Lipo-FITC-PAP-6 (FITC-LP) and DSPE-PEG-Pep@Lipo-FITC-PAP-6 (FITC-DPP), and the cellular uptake of FITC-DPP was qualitatively analyzed using an inverted fluorescence microscope. THP-1 cells in the logarithmic growth phase were taken and cultured at 3×10 4 Each well was inoculated in a 24-well plate, stimulated by adding phorbol ester, and cultured in an incubator for 12 hours. After the cells adhered to the wall, the culture medium was aspirated and washed three times with PBS. DSPE-PEG-Pep@Lipo-FITC-PAP-6 was prepared into a 50μM solution of PAP-6 using DMEM culture medium, and 0.5mL was added to each well, with three parallel wells in each group. The well plate was placed in an incubator and continued to be cultured. After 1 or 2 hours, it was taken out, the culture medium was aspirated, and washed three times with PBS. 300μL of cell fixative was added to each well for 15 minutes, the fixative was aspirated and washed three times with PBS, 300μL of DAPI was added to each well, stained for 15 minutes, the stain was aspirated and washed three times with PBS, and then observed under a fluorescence microscope. According to the above operation, the cells were taken out after being cultured for 1 or 2 hours, the culture medium was aspirated, the cells were washed three times with PBS, digested with trypsin for 2 minutes, washed three times with PBS, resuspended in 300 μL PBS, filtered through a 300-mesh filter, and analyzed by flow cytometry.

[0086] In vitro release study of DSPE-PEG-Pep@Lipo-PAP-6

[0087] 2 mL of DSPE-PEG-Pep@Lipo-PAP-6 solution of equal concentration (CPAP-6 = 0.40 mg / mL) was precisely drawn into a pre-treated dialysis bag with a molecular weight cutoff of 100 KD, and immersed in 20 mL of release medium (1% Tween 80 pH 7.4 PBS solution), respectively. The release medium temperature was 37°C and the speed was 100 rpm. 0.2 mL of release medium was precisely drawn at 0, 0.5, 1, 2, 4, 8, 12, and 24 h, methanol was added, and ultrasonic was performed for 15 min. After filtering through a 0.22 μm filter membrane, the supernatant was taken for HPLC testing. At the same time, the release medium of equal volume was added at the same temperature, and the test was repeated 3 times. The cumulative release rate of PAP-6 at each time point is calculated as follows: the cumulative release rate at each time point Q = Qn + (C1 + ... Ci-1 + Ci) × V1 / V2 × 100%, where Ci represents the PAP-6 concentration measured at different time points, V1 represents the fixed sampling volume at each time point, V2 represents the release medium volume, Qn represents the PAP-6 dissolution amount measured at each time point, and each sample is measured 3 times to calculate the average value. The in vitro release curve is drawn with the sampling time as the abscissa and the cumulative release rate of the drug as the ordinate, and the release curve is fitted using the zero-order equation, the first-order equation and the Higuchi equation.

[0088] Study on in situ retention of DSPE-PEG-Pep@Lipo-PAP-6 in colon

[0089] Small animal active imaging was used to characterize the in situ residence of DSPE-PEG-Pep@Lipo-PAP-6 in the colonic mucosa. PAP-6 peptide was labeled with Cy7-NHS, and DSPE-PEG-Pep@Lipo-Cy7-PAP-6 solution was prepared. After rectal perfusion of UC mice, 100 μL was administered, and the mice were detected by an in vivo imaging system (IVISVRLumina II) at 4, 24, and 48 h. The fluorescence signal of regional fluorescence was analyzed using the software of the in vivo imaging system. After the last photo was taken, the mice were killed by cervical dislocation, and the mouse colon was quickly removed and observed and photographed using the in vivo imaging system. PAP-6 peptide was labeled with FITC and DSPE-PEG-Pep@Lipo-FITC-PAP-6 solution was prepared. 100 μL was given by rectal perfusion to UC mice. The mice were killed by cervical dislocation at 4, 24, and 48 hours. The colon was quickly removed and the sample was taken 4 cm away from the anus. The sample was immediately frozen and embedded. Frozen sections were made. The fluorescence signal was observed and photographed using a fluorescence microscope imaging system.

[0090] result

[0091] The particle size and potential test results of DSPE-PEG-Pep@Lipo-PAP-6 show that the particle size of liposomes incubated with different addition amounts has no significant change compared with that before incubation, but the Zeta potential gradually increases with the increase of addition amount. According to the ROS responsive aggregation test results of DSPE-PEG-Pep@Lipo-PAP-6, when the addition amount of DSPE-PEG-Pep exceeds 5% under the condition of 100μM, it can significantly promote the cross-linking of functional materials on the surface of liposomes, further form liposome aggregates, and increase the particle size. Therefore, the addition amount of the functional carrier material DSPE-PEG-Pep is selected to be 5% ( Figure 6 )

[0092] Particle size, potential and micromorphology

[0093] The average particle size of DSPE-PEG-Pep@Lipo-PAP-6 is 209.6±0.9nm, and the potential is -9.32±0.45mV, indicating that the particle size of liposomes does not change much after modification with DSPE-PEG-Pep, but the surface potential increases slightly. Transmission electron microscopy observed that DSPE-PEG-Pep@Lipo-PAP-6 has a spherical structure.

[0094] 4.7.2 ROS-responsive aggregation

[0095] After adding hydrogen peroxide solution with a final concentration of 100 μM to DSPE-PEG-Pep@Lipo-PAP-6, the liposome particle size gradually increased with time, and the particle size increase was most significant when the reaction lasted for 2 hours, indicating that the functional carrier material can quickly respond to cross-linking in the ROS environment. Transmission electron microscopy showed that multiple small-sized liposomes aggregated or piled up together. This may be because the functional carrier on the surface of DSPE-PEG-Pep@Lipo-PAP-6 was cross-linked in response to ROS, which prompted the small-sized liposomes to aggregate together to form liposome aggregates, while the larger-sized liposomes were difficult to aggregate together quickly due to steric hindrance ( Figure 7 ).

[0096] 4.7.3 In vitro uptake experiments

[0097] The blue fluorescence is the cell nucleus stained with DAPI, and the green fluorescence is the fluorescence of FITC. The results after Merge can be used to observe the fluorescence intensity 1 hour after administration. There is obvious green fluorescence on the cell membrane of the FITC group, Lipo-FITC-PAP-6 group and DSPE-PEG-Pep@Lipo-FITC-PAP-6 group, while the fluorescence of the FITC-PAP-6 group is not obvious, indicating that the phospholipid bilayer of the liposome is easier to approach the cell surface than PAP-6; the green fluorescence intensity of each group increased after 2 hours of administration, among which the FITC group and Lipo-FITC-PAP-6 group were the most obvious, while the fluorescence intensity of the liposome modified with DSPE-PEG-Pep was slightly weaker than that of the unmodified one. This may be because the modification with PEG enhances the hydrophilic force of the liposome, and the functional polypeptides are stimulated to cross-link and aggregate in the culture medium by the peroxides released by the cells, reducing the chance of contact with the cell surface, thereby reducing the cell uptake of the modified liposomes. Flow cytometry analysis showed that both liposomes and modified liposomes could significantly increase the relative expression of FITC in cells, and the relative uptake at 2h was more obvious than that at 1h. However, there was no significant difference between liposomes and modified liposomes, indicating that the modification of the carrier material did not significantly affect the relative uptake of liposomes by cells ( Figure 8 ).

[0098] 4.8 In vitro release results of DSPE-PEG-Pep@Lipo-PAP-6

[0099] According to the release amount of PAP-6, compared with the PAP-6 group, the Lipo-PAP-6 and DSPE-PEG-Pep@Lipo-PAP-6 groups can release drugs slowly and continuously, but there is no significant difference in the release rate of the two liposomes. The cumulative release amount reached more than 60% at 24 hours. This shows that DSPE-PEG-Pep modification cannot delay the rate of liposome drug release ( Fig. 9 ). The drug release amounts at each time point in the Lipo-PAP-6 group and the DSPE-PEG-Pep@Lipo-PAP-6 group were fitted with three different release models with the corresponding time. The results showed that the Higuchi release model was the model with the fitting R2 value closest to 1.

[0100]

[0101] 4.9 Results of DSPE-PEG-Pep@Lipo-PAP-6 in situ retention in the colon

[0102] After enema of colitis mice, the distribution of Cy7-PAP-6 in the mice at different time points was detected by small animal in vivo imaging. Within 4 hours after enema, each group had obvious fluorescence intensity in the colon and abdominal cavity, among which the DSPE-PEG-Pep@Lipo-Cy7-PAP-6 group had the strongest fluorescence, indicating that the functionalized liposomes could quickly penetrate into the tissue and responsively cross-link to form aggregates; as time extended to 24 hours, the Cy-7-PAP-6 drug further penetrated into the abdominal cavity and blood circulation system, and obvious fluorescence was observed in the liver; but at 48 hours, the fluorescence of the Cy7-PAP-6 group disappeared, the fluorescence intensity of the Lipo-Cy7-PAP-6 group decreased significantly, and the colon of the DSPE-PEG-Pep@Lipo-Cy7-PAP-6 group still had a certain intensity of fluorescence. By observing the isolated colon of mice, it can be seen that a strong fluorescence intensity can still be observed in the colon of the DSPE-PEG-Pep@Lipo-Cy7-PAP-6 group 48 hours after enema, indicating that the constructed delivery system has the function of prolonging the long-term residence of liposomes in the colon, which also provides guidance for the frequency of drug administration in subsequent efficacy experiments.

[0103] The distribution of FTIC-PAP-6 in the mouse intestine was observed in frozen sections using a fluorescence microscope. There was obvious fluorescence intensity in the colon tissue of each group 4 hours after enema. The FITC-PAP-6 group had obvious expression in the intestinal cavity and colon epithelial tissue, while the Lipo-FITC-PAP-6 and DSPE-PEG-Pep@Lipo-FITC-PAP-6 groups mainly penetrated into the colon mucosal tissue and inflammatory lesions. As time extended to 24 hours, there was only very little fluorescence in the intestine of the FITC-PAP-6 group, the number of Lipo-FITC-PAP-6 group penetrating into the mucosal tissue decreased, but the number penetrating into the serosal layer increased. The liposomes may have entered the systemic circulation through the blood vessels, while the fluorescence of the DSPE-PEG-Pep@Lipo-FITC-PAP-6 group in the colon mucosal tissue was still clearly visible. At 48 hours, the fluorescence of the FITC-PAP-6 group disappeared, and the fluorescence intensity of the Lipo-FITC-PAP-6 group decreased significantly, while the colon of the DSPE-PEG-Pep@Lipo-PAP-6 group still had a certain intensity of fluorescence. This indicates that the constructed delivery system has the function of rapid penetration and prolonged residence of liposomes in colon mucosal tissue ( Fig.10 ).

[0104] Test example: The efficacy of long-acting liposomes of cockroach polypeptide against UC mice

[0105] Establishment of UC model

[0106] After 7 days of adaptive feeding of Balb / c mice, except for the normal control group which was given pure water, the other mice were given DSS aqueous solution to drink freely for 10 days, with the solution changed every 2 days. They were first fed with 3.0% (w / v) DSS concentration for 6 days, then changed to 2.0% (w / v) and continued to be fed for 6 days. After the modeling was completed, they were fed with pure water for 6 days, for a total of 18 days.

[0107] Grouping and drug administration

[0108] Six mice were randomly selected as the normal control group (Control group), and the remaining mice were modeled according to the above method and randomly divided into model control group (Model group), mesalamine group (Mesalamine group, 200 mg / kg), PAP-6 solution group (PAP-6 group, 2.0 mg / kg), Lipo-PAP-6 group (LP group, PAP-6: 2.0 mg / kg), DSPE-PEG-Pep@Lipo-PAP-61 group (DPP-1 group, PAP-6: 2.0 mg / kg), and DSPE-PEG-Pep@Lipo-PAP-62 group (DPP-2 group, PAP-6: 2.0 mg / kg) according to their initial body weight, with 6 mice in each group. Starting from the 7th day after modeling, each medication group was given corresponding drugs by enema at 0.1mL / 20g, and the Control and Model groups were given PBS at 0.1mL / 20g. The Control, Model, Mesalamine, PAP-6, and LP groups were given once a day, the DPP-1 group was given once every 2 days, and the DPP-2 group was given once every 4 days, for 12 consecutive days.

[0109] Index observation and detection methods

[0110] Observation of general status Observe and record the changes in weight, diet, autonomous activity and mental state of each group of mice during the experiment. Disease activity index (DAI) scoring refers to HoPanSham and other standards. The DAI score of mice is calculated daily according to the weight loss degree, fecal characteristics and fecal occult blood of mice to evaluate the disease activity. The scoring standard is shown in Table 1. DAI score = weight loss score + fecal characteristics score + occult blood score

[0111] Table 1 Disease Activity Index Score Table

[0112]

[0113] Note: Weight loss (%) = (weight at a certain time point after modeling - weight before modeling) / weight before modeling × 100%

[0114] Organ index: The mice were lightly anesthetized with isoflurane, and blood was taken from the eyeballs quickly, then the mice were killed by dislocation. The liver, spleen, and thymus were taken, the blood on the surface was wiped off, and the weight was weighed to calculate the organ index. Organ index = organ mass (mg) / mouse weight (g) 2.3.4 Scoring of colon length and mucosal damage index (CMDI) The colon was removed, and the length of the colon was measured and then longitudinally cut open along the edge of the mesentery, the feces was removed, and the colon was rinsed in PBS solution, and the water was wiped off with filter paper. Then the removed colon was spread on white paper, and the colon mucosal damage was observed with the naked eye according to the standards of Wangui Yu et al. (see Table 22 for details), and the score was given.

[0115] Table 2 Colon mucosal injury index (CMDI) scoring criteria

[0116]

[0117] Colon tissue cytokine determination: 2 cm was cut from the dissected colon near the anus and quickly placed in a -80°C low-temperature refrigerator for storage. When testing, the colon tissue was taken out, and after gradient freezing and thawing at -20°C and 4°C, a 10% (W / V) homogenate was prepared with pre-cooled PBS, centrifuged at 4°C, 3500r / min for 10 minutes, and the supernatant was taken. The contents of IFN-γ, IL-4, IL-10, iNOS, MCP-1, TGF-β1, MPO, EGF, IL-17A, and GM-CSF in the colon tissue were detected strictly according to the instructions of the relevant kits.

[0118] Colon pathological morphology observation (Histopathological score, HS) The remaining colon tissue was rolled up, fixed with 4% paraformaldehyde, dehydrated, paraffin-embedded, sliced, stained, and HE sections were prepared. Pathological sections were observed under an optical microscope, and colon HS scores were calculated according to EkstrōmGM and other standards.

[0119] Table 3 Histopathological scoring table

[0120]

[0121] Note: Total score = epithelial cell score + inflammatory cell infiltration score

[0122] Glycogen PAS staining to detect colonic mucin

[0123] After dewaxing, hydration, and distilled water immersion, the paraffin sections were dripped with periodic acid solution, placed at room temperature for 1 hour, rinsed with distilled water, dripped with Schiff's reagent, placed at 37°C for 20 minutes, rinsed with running water for 10 minutes, stained with hematoxylin for 1.5 minutes, differentiated with acid ethanol for 3 seconds, rinsed with running water for 10 minutes, blued, dehydrated step by step, transparentized with xylene, sealed with neutral gum, and observed and photographed under a microscope.

[0124] Immunohistochemical detection of colon mucosal barrier-related proteins

[0125] After antigen retrieval and serum blocking, paraffin colon sections were added with claudin-1, occludin, and ZO-1 primary antibodies (1:3000) and incubated overnight at 4°C; washed with PBS and dried, HRP-labeled secondary antibodies were added to cover the tissues and incubated at room temperature for 1 hour; DAB color development solution was used to develop the color, and the development time was controlled under a microscope. Hematoxylin counterstaining was performed for 3 minutes; dehydrated and sealed, and examined under a microscope. The positive area of ​​the sections was measured using Image-ProPlus6.0 software.

[0126] Statistical analysis methods

[0127] All data were statistically analyzed using SPSS26.0 software. The measurement data were expressed as mean ± standard deviation (x ± s). Continuous data that conformed to the normal distribution were analyzed using One-way ANOVA. The LSD test method was used for inter-group comparison. The data that did not conform to the normal distribution were tested using Tamhane's T2 method. Repeated measurement continuous data were analyzed using repeated measurement variance analysis. P < 0.05 or P < 0.01 indicated statistically significant differences.

[0128] 3.11 Effects of cockroach polypeptide long-acting liposomes on NF-κB / NLRP3 / Caspase-1 pathway

[0129] Weigh an appropriate amount of colon tissue from each group of the above efficacy experiment, add RIPA high-speed tissue / cell lysis buffer, PMSF (100×), protease / phosphorylase inhibitor (100×), and homogenize with an automatic high-speed tissue grinder. After homogenization, place it in an ice bath for 30 minutes, centrifuge it at 12000rpm4℃ for 10 minutes, and take the supernatant for quantification using a BCA kit. Mix the protein sample with the loading buffer thoroughly, denature it at 100℃ for 10 minutes, and separate it by constant voltage electrophoresis. Take out the gel and transfer it to a PVDF membrane activated by methanol at a constant flow rate. Add the primary antibody solution to the transferred membrane: NF-κB, p-NF-κBp65, NLRP3, Caspase-1 (dilution ratio 1:1000) and GAPDH (dilution ratio 1:2000); place it on a flat shaker and incubate it overnight at 4℃. The membrane was washed 3 times with TBST, 5 min / time; horseradish peroxidase-labeled IgG secondary antibody (dilution ratio 1:15000) was placed on a flat shaker and incubated at room temperature for 60 min, followed by ECL chemiluminescence detection. The exposure time was set to 30 s, and the results were imaged and photographed. ImageJ analysis software was used to perform grayscale analysis of the protein bands in the image (relative expression of the target protein = grayscale value of the target protein band / grayscale value of the internal reference protein band).

[0130] The efficacy of long-acting liposomes of cockroach polypeptide against UC in mice

[0131] Effects of DPP on the general condition of UC mice

[0132] The mice in the normal control group had normal diet and drinking water, smooth, white, and clean hair, good mental state, black, hard, ellipsoidal feces, and light yellow urine with normal color. After modeling, the mice ate significantly less, became thinner, had loose, dull hair, loose or bloody stools attached to the anus and tail, and their activity level was significantly reduced. After treatment with mesalazine, LP, DPP-1, and DPP-2, the mental state of the modeled mice improved significantly, the phenomenon of curling up decreased, the activity level increased, and the sticky and bloody stools at the anus disappeared. Although the mental state of the mice in the PAP-6 group improved, their activity level was low, and some mice still had loose stools attached to the anus.

[0133] Effects of DPP on DAI scores in UC mice

[0134] The results of Mauchly's sphericity test showed that the DAI score data of each group of mice from 0 to 18 days did not meet the sphericity assumption (P < 0.05), so the Green housc-Geisser method was used to correct the degrees of freedom and analyze the results. The results of repeated measures analysis of variance showed that: (1) Interaction (time*group): The interaction of mouse DAI scores was statistically significant (P < 0.01), indicating that the time factor had different effects on the DAI scores of mice in different groups. (2) Time effect: The time effect of mouse DAI scores was statistically significant (P < 0.01), indicating that the DAI scores of mice at different time points were different. (3) Inter-group effect: The inter-group effect of mouse DAI scores was statistically significant (P < 0.01), indicating that the DAI scores of mice in different groups were different. On the 18th day of the experiment, the drug treatment ended. Compared with the normal control group, the DAI scores of the model group and each drug treatment group were significantly increased (P < 0.01); compared with the model control group, the DAI scores of the mesalazine group, PAP-6 group, LP group, DPP-1 group, and DPP-2 group were significantly decreased (P < 0.01); compared with the mesalazine group, the DAI score of the PAP-6 group was significantly increased (P < 0.05 or P < 0.01), and there was no significant difference in the high-dose group (P> 0.05); compared with the PAP-6 group, the DAI score of the DPP-1 group was significantly decreased (P < 0.05) ( Fig.11 ).

[0135] Table 4 Analysis of variance of the effect of DPP on the changes in DAI scores of UC mice

[0136]

[0137] Note: Mauchly's sphericity test a: P = 0.000; Greenhousc-Geisse correction b :P-0.415

[0138] Effects of DPP on organ indexes of UC mice

[0139] Compared with the normal control group, the thymus index of the model control group was significantly decreased (P < 0.01), and the spleen index was significantly increased (P < 0.01). Compared with the model control group, the spleen index of the mesalazine group, LP group, DPP-1 group and DPP-2 group was significantly decreased (P < 0.05 or P < 0.01), but only the mesalazine group and DPP-1 group could increase the thymus index (P < 0.05 or P < 0.01). Compared with the mesalazine group, the spleen index of the PAP-6 group and LP group was significantly decreased (P < 0.05 or P < 0.01). Compared with the PAP-6 group, the thymus index of the DPP-1 group was significantly increased (P < 0.05), and the spleen index was significantly decreased (P < 0.05) ( Fig.12 ). Effects of DPP on colon-related parameters in UC mice

[0140] The colon length of mice in the normal control group was longer, and there was no congestion, edema, erosion, or ulceration in the mucosa; the colon of mice in the model control group was shortened, and congestion and edema were visible in the intestinal wall, with the most severe lesions in the colon near the anus; the colon length and mucosal lesions of mice in each drug treatment group were improved. Compared with the normal control group, the colon length of mice in the model control group was significantly shortened (P < 0.01), and the CMDI score was significantly increased (P < 0.01); compared with the model control group, except for the PAP-6 group, the colon length of the other groups increased significantly (P < 0.05 or P < 0.01), and the CMDI score was significantly reduced (P < 0.05 or P < 0.01), among which the regulatory effect of the mesalazine group, LP group, and DPP-1 group was the most obvious (P < 0.01) ( Fig.13 ).

[0141] Effects of DPP on cytokines in colon tissue of UC mice

[0142] Compared with the normal control group, the expression levels of inflammatory factors TNF-α, IL-6, IL-17A, and MCP-1 as well as the contents of inflammatory-related active enzymes MPO, iNOS, and MMP-2 in the colon tissue of mice in the model control group were significantly increased (P < 0.01), and the expression level of the anti-inflammatory cytokine IL-10 was significantly decreased (P < 0.01). Compared with the model control group, the PAP-6 group could significantly reduce the expression levels of inflammatory factors TNF-α and IL-6 and the content of inflammatory-related active enzyme MPO in colon tissue, and significantly increase the expression level of anti-inflammatory cytokine IL-10 (P<0.01); the mesalazine group, LP group, DPP-1 group and DPP-2 group could effectively inhibit the expression levels of TNF-α, IL-6, IL-17A and MCP-1 (P<0.01) and the content of iNOS and MMP-2 (P<0.05 or P<0.01), and significantly increase the expression level of anti-inflammatory cytokine IL-10 (P<0.01); the pharmacodynamic effects of the LP group and the DPP-1 group were similar to those of the mesalazine group, while the pharmacodynamic effect of the DPP-2 group was inferior to that of the other three groups ( Fig.14 ).

[0143] Effects of DPP on colon HS scores in UC mice

[0144] Microscopic observation of colon pathological sections revealed that the colon structure of the normal control group was intact and clear, the glands were neatly arranged, obvious crypts and goblet cells were visible, and there was basically no congestion, edema, or inflammatory cell infiltration. The surface layer of the colon mucosa of mice in the model control group was sloughed and defective, the crypts and goblet cells were lost on a large scale, and a large number of inflammatory cells infiltrated. The goblet cells and crypts of the colon of mice in each treatment group showed varying degrees of repair and improvement, and inflammatory cell infiltration decreased. Compared with the normal control group, the epithelial cell score, inflammatory cell infiltration score and HS total score of the model control group were significantly increased (P < 0.01); compared with the model control group, each drug treatment group significantly reduced the epithelial cell score and HS total score (P < 0.01), and the inflammatory cell infiltration score was significantly reduced in the LP group and the DPP-1 group (P < 0.01), among which the DPP-1 group had the most significant reduction in the HS total score ( Fig.15 ).

[0145] Effect of DPP on colon mucin production in UC mice

[0146] Mucin is a mucus colloid covering the intestinal mucosal epithelial cells. Its molecular structure is a macromolecular glycoprotein mainly secreted by goblet cells. When goblet cells are stimulated by inflammation, they will promote the excessive excretion of immature thin mucus, weakening the barrier protection of the mucus layer. The goblet cells in the normal control group had normal morphology and secreted more mucin in the glandular cavity, while the expression and distribution of mucin in the model control group were significantly lower than those in the normal group. After treatment with each drug group, the expression and distribution of mucin in the goblet cells of the mouse colon increased significantly. Except for the PAP-6 group, the expression and distribution of mucin in the goblet cells of the other groups increased significantly compared with the model control group.

[0147] Effects of DPP on the production of tight junction proteins in colonic mucosa of UC mice

[0148] Claudin, occludin-1 and ZO-1 are key proteins of the tight junctions of intestinal epithelial cells in the mechanical barrier of the colon. Under inflammation, the expression of tight junction proteins decreases, resulting in increased permeability of the intercellular space, causing antigens to enter the intestine and aggravate local inflammation. Compared with the normal control group, the protein expressions of claudin-1, occludin and ZO-1 in the model control group were significantly reduced (P<0.01). Compared with the model control group, except for the PAP-6 group, which failed to promote the expression of occludin protein, the other treatment groups could effectively promote the expression of the three tight junction proteins (P<0.05 or P<0.01), among which the DPP-1 group had the most obvious promoting effect ( Fig.16 ).

[0149] Effects of cockroach polypeptide long-acting liposomes on NF-κB / NLRP3 / Caspase-1 pathway

[0150] More and more studies have shown that the overactivation of immune cells is related to the pathogenesis of UC. Polarization of macrophage M1 can enhance the signal transduction of NF-κB and produce a large number of inflammatory factors such as TNF-α, IL-1β, IL-6, etc. At the same time, p-NF-κBp65 further promotes the inflammasome formed by NLRP3 assembly, turning the inactive cysteine ​​protease precursor into activated caspase-1, and cleaving the N-terminal sequence of glucosamine D (GSDMD), allowing it to bind to the membrane to produce membrane pores, leading to macrophage pyroptosis and releasing a large amount of IL-1β and IL-18 into the body, aggravating local inflammation. According to the results of WB experiments, compared with the normal control group, the expression levels of NLRP3, NF-κB, p-NF-κBp65, and Caspase-1 proteins in the colon tissue of mice in the model control group were significantly increased (P < 0.05); after treatment with each drug group, it was found that compared with the model control group, except for the PAP-6 group, which failed to significantly improve the expression of the above four proteins, the other treatment groups could effectively inhibit the expression levels of the four proteins (P < 0.05 or P < 0.01). Among them, the inhibitory effect of the DPP-1 group was close to that of the positive drug ( Fig.17 ).

[0151] discuss

[0152] Although conventional liposome dosage forms can effectively improve the local permeation of drugs in the colon, they still cannot effectively solve the problem of poor patient compliance caused by frequent clinical administration. According to the large amount of ROS in the inflammatory microenvironment, a functional carrier material with ROS response cross-linking was innovatively designed to construct colon in situ long-acting liposomes with inflammatory microenvironment response aggregation, and PAP-6 peptide was used as a model drug for research, and the mechanism of action of the in situ long-acting liposome against UC was preliminarily explored. Through the study of the physicochemical properties of PAP-6 peptide, it was found that it has strong water solubility and is insoluble in dichloromethane, methanol, and acetonitrile, but it can be completely dissolved in a certain proportion of methanol solution. Therefore, when preparing liposomes, the peptide can only be dissolved in the aqueous phase for hydration preparation. At the same time, by examining the effect of different methanol ratios on the recovery rate of PAP-6, the demulsification conditions of the liposomes were further determined to be 30% methanol and 15 minutes of ultrasound. In order to avoid the interference of free drugs on the activity of Lipo-PAP-6, the free drugs in the liposome solution were removed by dialysis, and the functional carrier material was modified to the surface of the liposome by incubation. In view of the idea that ROS highly expressed in colon lesions can induce thiol cross-linking to form disulfide bonds, the structure of MMP-2 sensitive peptide was optimized to increase its hydrophilicity and cross-linking function, and inserted into the liposome surface with the help of DSPE-PEG. In vitro studies have found that the increase in the hydrophilicity of the peptide is conducive to the cross-linking of the functional carrier. In vitro 100μM hydrogen peroxide solution, the particle size of the liposome modified with the functional carrier material increased significantly. At the same time, the particle size and potential of the liposome did not change significantly before and after modification, but TEM showed that the modified liposome was more round. According to the results of in vitro release of peptides by DSPE-PEG-Pep@Lipo-PAP-6, the Higuchi release model had the highest fitting degree, indicating that the drug release characteristics are a combination of dissolution and diffusion, indicating that the functionalized liposomes quickly penetrate into the lesion site, aggregate after ROS responsive cross-linking, and then slowly release the drug to be absorbed by the colon tissue. Through the colon in situ residence experiment, it was found that the drug was still retained in the colon of UC mice 2 days after administration, indicating that the functionalized liposomes can prolong the residence time of the drug in the lesion site, which also provides an experimental basis for the administration cycle of subsequent efficacy experiments. According to the efficacy study of DSPE-PEG-Pep@Lipo-PAP-6, it was found that the delivery system can effectively improve multiple inflammatory indicators of model mice when administered once / 2d or once / 4d, and there is no significant difference compared with the Lipo-PAP-6 liposomes with unmodified functional carrier materials administered once / 1d, but multiple indicators are better than the experimental group administered once / 1d, which shows that the constructed PAP-6 colon in situ long-acting liposomes can effectively improve the problem of short in situ residence time after enema and the need for frequent administration, which has guiding significance for the development of subsequent products and clinical applications.

Claims

1. An inflammatory microenvironment-responsive cross-linked polypeptide-modified liposome DSPE-PEG-Pep@Lipo-PAP-6, comprising: Drug, soybean lecithin, cholesterol and DSPE-PEG-Pep B, wherein the drug, soybean lecithin and cholesterol form liposome Lipo-PAP-6, DSPE-PEG-Pep B is modified on the surface of the liposome, the DSPE-PEG-Pep B is obtained by the reaction of DSPE-PEG-NHS and Pep B, and the amino acid sequence of Pep B is PLGVRGGGGC.

2. The liposome DSPE-PEG-Pep@Lipo-PAP-6 according to claim 1, characterized in that: The drug is selected from active ingredients with anti-colitis effects, selected from mesalazine, sulfasalazine, racecadotril, sodium para-aminosalicylate, dexamethasone, methylprednisolone, azathioprine or PAP-6.

3. The liposome DSPE-PEG-Pep@Lipo-PAP-6 according to claim 1, characterized in that: In the liposome Lipo-PAP-6, the mass ratio of drug, soybean lecithin and cholesterol is 0.635:2.5:12.

5.

4. The liposome DSPE-PEG-Pep@Lipo-PAP-6 according to claim 1, characterized in that: The mass ratio of the DSPE-PEG-Pep B to Lipo-PAP-6 is 1:

20.

5. The method for preparing the liposome DSPE-PEG-Pep@Lipo-PAP-6 modified with the inflammatory microenvironment responsive cross-linked polypeptide according to claim 1, comprising the following steps: 1) Preparation of DSPE-PEG-Pep B: Weigh DSPE-PEG-NHS and Pep B, dissolve in PBS, stir at room temperature in the dark, take out the reaction solution, dialyze in distilled water and freeze-dry; 2) Preparation of Lipo-PAP-6: weigh soybean lecithin and cholesterol, add dichloromethane to dissolve them, evaporate and remove dichloromethane to form a lipid film, weigh PAP-6, prepare a solution with PBS, pour it into the lipid film, hydrate it with constant temperature oscillation to obtain a preliminary product of Lipo-PAP-6, extrude the preliminary product through a liposome extruder with a microporous filter membrane to obtain Lipo-PAP-6; 3) Preparation of DSPE-PEG-Pep@Lipo-PAP-6: Take Lipo-PAP-6, add DSPE-PEG-Pep B in proportion, incubate for 4 hours, and prepare DSPE-PEG-Pep@Lipo-PAP-6.

6. The preparation method according to claim 5, characterized in that: In the step 1), the molar ratio of DSPE-PEG-NHS to Pep is 1.2:

1.

7. The preparation method according to claim 5, characterized in that: The hydration condition in the step 2) is to hydrate the mixture at 30° C. and 200 rpm in a constant temperature oscillator for 0.75 h.

8. The preparation method according to claim 5, characterized in that: The incubation condition in step 3) is 37° C. for 4 h.

9. Use of the liposome DSPE-PEG-Pep@Lipo-PAP-6 modified with the inflammatory microenvironment responsive cross-linked polypeptide according to claim 1 in the preparation of a drug for treating colitis.

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