Cathepsin B-reduction double-response type biological self-degradation cationic lipid as well as preparation and application of lipid nanoparticles of cathepsin B-reduction double-response type biological self-degradation cationic lipid

Abstract

The invention discloses cathepsin B-reduction dual-response type biological self-degradation cationic lipid and preparation and application of lipid nanoparticles thereof, belongs to the technical field of new auxiliary materials and new dosage forms of pharmaceutical preparations, and relates to a preparation method of cathepsin B and reduction dual-response type self-degradation cationic lipid and construction of siRNA lipid nanoparticles of the cathepsin B and reduction dual-response type self-degradation cationic lipid. The invention discloses colonizable inulin-spirulina composite gel for colon targeted delivery and application of the colonizable inulin-spirulina composite gel in nucleic acid drug delivery. According to the invention, a colon-specific modular hydrogel platform is designed by integrating a microalgae biotechnology and a nano-drug, and a new strategy and choice are provided for solving the toxic and side effects of lipid nanoparticles and oral delivery of RNA (Ribonucleic Acid).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of new excipients and new dosage forms of pharmaceutical preparations, and relates to the design and synthesis of cathepsin B-reducing dual-responsive biodegradable cationic lipids, the preparation of biodegradable lipid nanoparticles by wrapping siRNA under the action of auxiliary lipids, cholesterol and polyethylene glycol lipids, and the preparation of composite gels with spirulina and inulin, and the application in treating ulcerative colitis related diseases. BACKGROUND

[0002] Inflammatory bowel disease (IBD) mainly includes ulcerative colitis (UC) and Crohn's disease (CD), which is a chronic inflammatory disease. UC is difficult to cure, and the risk of colon cancer increases with the progression of inflammation, often requiring surgery and lifelong medication. Small molecule non-targeted therapy may cause systemic adverse reactions and side effects. siRNA can precisely regulate the expression of IBD related pro-inflammatory cytokines, effectively relieving the progression of IBD. Since the main pathological changes of UC occur in the intestinal tissue, oral administration is considered the best way to directly deliver drugs to the pathogenic site in the intestine. However, the oral administration of biological preparations, especially RNA drugs, is hindered by the susceptibility of the gastrointestinal environment and gastrointestinal emptying. In addition, the hydrophilic and negatively charged nature of nucleic acids hinders their cellular uptake and escape from lysosomes to the cytoplasm to exert drug efficacy. In view of the cell barriers and gastrointestinal environment obstacles faced by oral delivery of nucleic acid drugs, the present inventors propose a solution: design a cationic lipid sensitive to both cathepsin B (CTSB) and glutathione (GSH) to prepare lipid nanoparticle loaded siRNA drugs. The electrostatic force is used to construct a spirulina-lipid nanoparticle micro-nano integrated system, which is further mixed with inulin gel (IG) to prepare an oral nucleic acid drug delivery carrier with colon targeting. Specifically, phenylalanine-lysine (Phe-Lys) is a dipeptide fragment sensitive to CTSB, and lysine adsorbs siRNA through positive electricity, and the dual sensitivity of CTSB and GSH can increase the on-demand release of drugs. IG is specifically degraded by the flora in the colon, which can protect the drugs from the damage of gastrointestinal juices and digestive enzymes in the upper digestive tract, and endows the preparation with colon targeting. In order to further increase the colon colonization ability of the nanoparticles, the lipid nanoparticles are adsorbed on the surface of spirulina through electrostatic force, and the spiral structure can be captured by intestinal villi to increase the residence time of the drugs. At the same time, spirulina and inulin as natural prebiotics play a role in regulating intestinal flora, which plays a synergistic role in siRNA treatment of colitis. SUMMARY

[0003] The technical problem solved by the present application is to overcome the defects of poor biodegradability and lysosome escape efficiency of existing lipid nanoparticles, improve the colon-targeted delivery efficiency of the lipid nanoparticles and increase the intestinal colonization efficiency of the lipid nanoparticles, and design a composite inulin gel with good biological safety and high siRNA delivery efficiency.

[0004] The purpose of the present application is to design and synthesize biodegradable cationic lipids containing disulfide bonds and cathepsin B-sensitive in vivo, prepare a composite inulin gel encapsulating siRNA lipid nanoparticles, and its treatment in ulcerative colitis. The cytotoxicity of the lipid nanoparticles and the therapeutic effect of the composite gel on colitis in mice are investigated to provide new strategies and more choices for the development of gene therapy for colitis and meet the urgent needs of safe and efficient lipid nanoparticles in clinical practice.

[0005] The present application achieves the above-mentioned purpose by the following technical solutions: In a first aspect, the present application provides a biodegradable cationic lipid under intracellular high reduction and high cathepsin B conditions, which is obtained by amide reaction of a fatty acid carbon chain containing a cathepsin B-sensitive dipeptide fragment bridged by a disulfide bond.

[0006] The cathepsin B-sensitive fatty acid carbon chain is N-[(S)-1-[[(S)-6-amino-1-oxohexyl-2-yl]amino]-1-oxo-3-phenylpropyl-2-yl]tetradecanamide.

[0007] Further, the N-[(S)-1-[[(S)-6-amino-1-oxohexyl-2-yl]amino]-1-oxo-3-phenylpropyl-2-yl]tetradecanamide prepared by the present application is reacted with 2,2'-dithiodiethylamine to obtain a biodegradable cationic lipid.

[0008] The biodegradable cationic lipid according to the present application has the following structural formula: .

[0009] In a second aspect, the present application provides a preparation method of the biodegradable cationic lipid according to the first aspect, and the specific synthesis route and method are as follows: The compd 1 (2 ep) was dissolved in 10 mL DMF, cooled to 0°C, then added EDCI (2 eq), HOOBt (2 eq), 0°C for 10 min, then added compd 2 (1 eq), NMM (3.3 eq), transferred to room temperature for 2 h, LC-MS monitoring reaction complete, spin off the solvent, then added 10 mL TFA, reacted at room temperature for 2 h, added 100 mL ether, a large amount of solid precipitated, centrifuged and dried, to obtain the FK crude product.

[0010] The obtained crude product was dissolved in acetonitrile: water (8:2) and filtered through a 0.45 μM microporous filter, and then injected into a preparative liquid phase for separation and purification, to obtain the target product.

[0011] Chromatographic conditions: Chromatographic column: octadecylsilane bonded silica gel as filler (YMC-Pack ODS-A 250*20 mm l.D., S-5 μm 12 nm); Detection wavelength: 220 nm; Mobile phase: acetonitrile: water = 80:20; Retention time: 45 min.

[0012] In a third aspect, the present application further provides a biodegradable lipid nanoparticle, which comprises the cathepsin B-reduction dual-responsive biodegradable cationic lipid and siRNA described above.

[0013] In a fourth aspect, the present application further provides an oral microalgae-in-nanomodular integrated gel system, which comprises spirulina and inulin.

[0014] Preferably, the spirulina and the lipid nanoparticle described above are obtained by electrostatic interaction to form a microalgae-in-nanomodule, and then a microalgae-in-nanomodular integrated gel system is obtained by heating and cooling.

[0015] In a fifth aspect, the present application further provides the use of the cathepsin B-reduction dual-responsive biodegradable cationic lipid or the lipid nanoparticle or the gel system comprising the same described above in the preparation of a nucleic acid drug delivery carrier.

[0016] Preferably, the nucleic acid drug delivery carrier is used for oral administration.

[0017] In a sixth aspect, the present application further provides the use of the cathepsin B-reduction dual-responsive biodegradable cationic lipid or the lipid nanoparticle or the gel system comprising the same described above in the preparation of a drug for treating intestinal diseases.

[0018] As an optional mode, the drug is used for ulcerative colitis or Crohn's disease.

[0019] The present application has the following beneficial effects: The preparation of the lipid nanoparticle by the cationic lipid increases the siRNA stability, improves the lysosome escape efficiency of the lipid nanoparticle; the disulfide bond and cathepsin B sensitive dipeptide fragment are introduced into the cationic lipid, which promotes the degradation of the lipid nanoparticle under the action of glutathione (GSH) and cathepsin B in the cytoplasm, realizes the rapid release of siRNA in the cell, improves the gene delivery efficiency and reduces the toxic side effects in the body. On this basis, the spirulina is designed to improve the colonization capacity of the nanoparticle in the intestinal tract, and the inositol gel is wrapped to realize the targeted delivery in the colon. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The H-NMR spectrum of the biodegradable cationic lipid FK in the embodiment 1 of the present application. 1 H‐NMR spectrum.

[0021] Figure 2 The high-resolution mass spectrum of the biodegradable cationic lipid FK in the embodiment 1 of the present application.

[0022] Figure 3 The particle size distribution graph and the transmission electron microscope graph of the cationic lipid nanoparticle FK LNPs@siCy5 in the embodiment 2 of the present application.

[0023] Figure 4 The in vitro release graph of the biodegradable cationic lipid nanoparticle in the embodiment 3 of the present application under non-reducing conditions, reducing conditions and cathepsin B conditions.

[0024] Figure 5 The hemolysis graph of the biodegradable cationic lipid nanoparticle in the embodiment 3 of the present application under different pH conditions. Wherein, A: hemolysis pictures of the lipid nanoparticle with different concentrations under pH 5.0 and pH 7.4 conditions; B: hemolysis degree graph of the lipid nanoparticle with different concentrations under pH 5.0 and pH 7.4 conditions.

[0025] Figure 6 The appearance and scanning electron microscope of the spirulina, spirulina-lipid nanoparticle and oral microalgae inner nanomodular integrated gel system in the embodiment 4 of the present application.

[0026] Figure 7 The gastrointestinal tract stability graph of the composite inositol gel in the embodiment 5 of the present application.

[0027] Figure 8 The tissue distribution graph of the inositol composite gel in the embodiment 6 of the present application, which is the fluorescence imaging graph of the gastrointestinal tract distribution of the preparations in each group.

[0028] Figure 9 The fluorescence semi-quantitative diagram of the tissue distribution of the composite inulin gel in Example 6 of the present application.

[0029] Figure 10 The pharmacodynamic experiment diagram of the composite inulin gel in Example 7 of the present application on mice with colitis. Among them, A: the colon pictures of mice in different treatment groups; B: experimental grouping; C: the body weight changes of mice in different treatment groups during the experiment; D: the DAI scores of mice in different treatment groups; E: the colon length quantification diagram of mice in different groups.

[0030] Figure 11 The levels of colitis-related factors in the serum of mice in the pharmacodynamic experiment treatment of the composite inulin gel in Example 7 of the present application on mice with colitis. Among them, A: the level of interleukin 1-β in the serum of mice; B: the level of tumor necrosis factor-α in the serum of mice; C: the level of interleukin-6 in the serum of mice; D: the level of transforming growth factor-β1 in the serum of mice; E: the level of myeloperoxidase in the serum of mice; F: the level of fluorescein isothiocyanate-dextran in the serum of mice.

[0031] Figure 12 The fluorescence section of the tight junction protein ZO-1 in the colon of mice in the pharmacodynamic experiment of the composite gel in Example 7 of the present application on mice with colitis.

[0032] Figure 13 The H&E staining of the colon of mice in different treatment groups in the pharmacodynamic experiment of the composite gel in Example 7 of the present application on mice with colitis.

[0033] Figure 14 The liver and kidney function indicators of mice in different treatment groups in the pharmacodynamic experiment of the composite gel in Example 7 of the present application on mice with colitis. Among them, A: the level of aspartate aminotransferase (AST) in the serum of mice; B: the level of alanine aminotransferase (ALT) in the serum of mice; C: the level of blood urea nitrogen (BUN) in the serum of mice; D: the level of creatinine (CRE) in the serum of mice.

[0034] Figure 15 The influence of the composite gel in Example 8 of the present application on the α diversity of the intestinal flora of mice in the pharmacodynamic experiment on mice with colitis. Among them, A: observed species (Observed species); B: Chao 1 index; C: Shannon index; D: Simpson index.

[0035] Figure 16 The β diversity analysis of the intestinal flora of mice in the pharmacodynamic experiment of the composite gel in Example 8 of the present application on mice with colitis.

[0036] Figure 17 The influence of the composite gel in Example 8 of the present application on the composition of intestinal flora species in mice in the pharmacodynamic experiment of mice with colitis. DETAILED DESCRIPTION

[0037] The above content of the present application is further described in detail by the following specific examples, but does not represent the limitation of the embodiments to the present application.

[0038] Example 1: Synthesis of biodegradable “twin-like” cationic lipids with cathepsin B-reducible dual-responsive MA-Phe-Lys (2 ep) was dissolved in 10 mL DMF, cooled to 0°C, then EDCI (2 eq), HOOBt (2 eq) were added, reacted at 0°C for 10 min, then 2,2'-dithiodiethylamine (1 eq), NMM (3.3 eq) were added, and the reaction was transferred to room temperature for 2 h. LC-MS was used to monitor the completion of the reaction. The solvent was removed by spinning, 10 mL TFA was added, and the reaction was carried out at room temperature for 2 h. 100 mL of ether was added, a large amount of solid was precipitated, centrifuged and dried, and MA-FK-SS crude product was obtained.

[0039] Then the crude product was dissolved in acetonitrile: water (8:2) and filtered through a 0.45 μM microporous filter, and injected into a preparative liquid phase for separation and purification. The chromatographic conditions are as follows: Chromatographic column: octadecylsilane bonded silica gel as filler (YMC-Pack ODS-A 250*20 mm l.D., S-5 μm 12 nm); Detection wavelength: 220 nm; Mobile phase: acetonitrile: water = 80:20; Retention time: 45 min.

[0040] Finally, the target product was obtained, with a yield of about 50%.

[0041] The structure of MA-FK-SS in Example 1 was confirmed by high resolution mass spectrometry (HRMS) and nuclear magnetic resonance hydrogen spectrum. The results are shown in Figures 1-2 d-DMSO was selected as the solvent in nuclear magnetic resonance, and the spectrum analysis results are as follows: 1 H NMR (400 MHz, Methanol- d 4) δ 7.29 – 7.20 (m, 10H), 4.66 – 4.49 (m, 2H), 4.37 – 4.25 (m,2H), 3.51 – 3.42 (m, 4H), 2.93 – 2.88 (t, J = 6.8 Hz, 4H), 2.81 (t,J = 6.8Hz, 4H), 2.17 (t, J = 7.8 Hz, 4H), 1.68 - 1.60 (m, 4H), 1.51 - 1.45 (m, 4H), 1.35 - 1.17 (m, 52H), 0.90 (t, J = 6.9 Hz, 6H).ESI-MS:m / z = 1145.758140 [M+Na] + Example 2: Preparation of TNFα siRNA-loaded biodegradable ionizable lipid nanoparticles MA-FK-SS (2 mg), DSPC (2 mg), cholesterol (2 mg) and DMG-PEG2 K (2 mg) were precisely weighed into 1 mL anhydrous ethanol to obtain a stock solution of SS-MA / DSPC / cholesterol / DMG-PEG 2K with a concentration of 2 mg / mL; then, SS-MA, DSPC, cholesterol, DMG-PEG 2K were mixed uniformly at a molar ratio of 30:48.5:20:1.5 (oil phase); 10 ODcy5 siRNA was dissolved in 330 μL of citric acid-sodium citrate buffer (0.01 mol / L, aqueous phase); then the oil phase and the aqueous phase were mixed at a volume ratio of 1:3 and vortexed for 30 min, with the mass ratio of total lipids to TNFα siRNA being 20:1; after vortexing, ethanol was removed by ultrafiltration centrifugation to obtain FK LNPs@siCy5; the same method was used to prepare MC3 LNPs@siCy5 with D-Lin-MC3-DMA as a control.

[0042] The results are shown in Table 1, the particle size of the lipid nanoparticles is 67 nm, the polydispersity index (PDI) is about 0.2, and the encapsulation efficiency is about 96%. Figure 3 The transmission electron microscopy results show that FK LNPs@siCy5 has a spherical particle structure.

[0043] Table 1: Particle size and encapsulation efficiency of "twin-like" lipid nanoparticles Example 3: In vitro release test of biodegradable cationic lipid nanoparticles The release media are: a. PBS (pH 7.4), b. GSH (10 mM), 61.466 mg of GSH is dissolved in 20 mL of Mcllvaine buffer, c. Papain (100 U / mL), 5 mg of papain is dissolved in 20 mL of 10 mM GSH solution, GSH plays a role in activating the activity of papain in the papain release medium, with a double sensitive environment.

[0044] A certain amount of FK LNPs@siCy5 and MC3 LNPs@siCy5 is taken in 3 mL of release medium, and the equivalent dose of Cy5 siRNA in the release system is (0.625 μg / mL), and the release system is incubated at 37°C in a 100 rpm shaker. At the designated time point, 500 μL of the release system is centrifuged at 13000 rpm for 15 min, and the supernatant is taken to determine the fluorescence intensity of free Cy5 siRNA, which is substituted into the standard curve to calculate the in vitro release rate of Cy5 siRNA.

[0045] As shown in Figure 4 GSH and the like reducing substances are used to activate the activity of papain, and GSH and CTSB can simulate a double sensitive microenvironment. In PBS solution, the cumulative release amount of FK LNPs is about 20% within 24 h, under intracellular high glutathione concentration, the cumulative release amount of FK LNPs is about 40%, and under glutathione and CTSB conditions, the cumulative release amount of FK LNPs is about 70% within 24 h. The in vitro release results show that FK with double sensitivity increases the release amount of siRNA, and has good in vitro release behavior.

[0046] Rat orbital blood is collected in an anticoagulant tube, centrifuged at 500 g for 5 min, and the supernatant plasma component is discarded. Add physiological saline to wash the red blood cells gently several times until the supernatant is colorless. After discarding the supernatant, pure red blood cells are obtained, and a 2% red blood cell suspension is prepared in pH 5.0 and pH 7.4 buffers. Add lipid nanoparticle preparation to the 2% red blood cell suspension so that the final concentration of total lipid components in the lipid nanoparticle in each sample is 50 μg / mL. The positive control group adds the same volume of 20% TritonX-100 as the preparation. The negative control group adds the same volume of corresponding pH buffer, and mixes uniformly at 37°C in a shaker for 1 h. After incubation, the sample is centrifuged at 13000 rpm for 10 min, and the supernatant is aspirated and tested for hemoglobin absorbance at 540 nm wavelength using a microplate reader. The positive control is considered as 100% hemolysis, and the hemolytic ability of different concentrations of lipid nanoparticles at different pH conditions is calculated.

[0047] AsFigure 5 As shown in A, under the condition of pH 7.4, after incubation of FK LNPs or MC3 LNPs and red blood cells, most of the red blood cells were precipitated on the bottom of the EP tube, and the supernatant had no obvious red color; under the condition of pH 5.0, the supernatant of FK LNPs and MC3 LNPs was blood red, and this phenomenon was caused by the rupture of red blood cell membrane, indicating that under acidic conditions, the lipid nanoparticles had a certain destructive effect on the biological membrane, which was beneficial to the function of lysosome escape. The results of hemolysis rate quantification are as follows Figure 5 As shown in B, when the concentration of lipid nanoparticles was 50 μg / mL, the hemolysis rate of FK LNPs was 1.5 times that of MC3 LNPs. The above results show that the prepared FK LNPs have a hemolysis rate of <5% under physiological conditions, and will not destroy the normal biological membrane, and have good biological safety, and FK LNPs have a destructive effect on the biological membrane under acidic conditions, which may be beneficial to increase the ability of lysosome escape.

[0048] Example 4: Preparation of oral microalgae-in-nanomodular integrated gel system The cultured spirulina (SP) was collected and washed with pure water three times before use. Freeze-drying was carried out. 50 mg of SP was resuspended in 1 mL of pure water to prepare a SP suspension. FK LNPs and SP suspension were incubated at room temperature for 30 min, and after incubation, the precipitate was collected by centrifugation at 4500 rpm for 10 min, which was the prepared SP-FK LNPs. The supernatant was used to determine the fluorescence signal of Cy5 siRNA, and the concentration of free FK LNPs was calculated according to the standard curve. 500 mg of inulin was fully dissolved in 1 mL of pure water, heated at 70°C for 5 min, and quickly cooled to room temperature in ice water. The prepared SP-FK LNPs were added to the inulin solution and mixed quickly, and the IG@SP-FK LNPs were prepared by continuously shaking and cooling to gel state.

[0049] As shown in Figure 6 , the appearance of blank SP was green, and after incubation with FK LNPs@siCy5, the preparation turned blue-green; the IG@SP-FK LNPs remained at the bottom of the bottle after being inverted, proving that the preparation had good adhesion. The results of scanning electron microscopy showed that the blank SP was slightly shrunk due to dehydration, but the surface was still smooth; after incubation of SP and FK LNPs, the surface of SP-FK LNPs had granular protrusions, indicating that a large number of FK LNPs were attached to the surface of SP. The scanning electron microscopy of IG@SP-FK LNPs showed no spiral structure in the field of view, mainly showing the appearance of inulin, proving the successful coating of inulin.

[0050] Example 5: Gastrointestinal stability experiment of oral microalgae-in-nanomodular integrated gel system A mouse colitis model was established by dissolving sodium dextran sulfate (DSS) at a concentration of 3% in water and allowing mice to drink the 3% DSS solution freely for 6 days. C57BL / 6 mice were fasted for 12 hours beforehand and orally administered 500 mL of IG@SP-FKLNPs. Gastric, small intestinal, and colonic contents were collected at 0.5 h, 4 h, and 6 h, respectively. The intestinal contents were freeze-dried, and the morphology of the formulation was observed using scanning electron microscopy.

[0051] like Figure 7 As shown, in the stomach and small intestine, the intestinal contents exhibit the appearance of inulin, indicating that the inulin gel remains stable in the upper gastrointestinal tract. Electron microscopy of the colonic contents reveals partially exposed SP-FK LNPs micro / nanosystems from the inulin gel, with an incompletely degraded inulin layer visible on the SP-FK LNP surface. These gastrointestinal stability results suggest that the inulin gel protects the micro / nanosystems from the acidic environment of the stomach and gradually degrades in the colon, thus achieving colon-targeting.

[0052] Example 6: Tissue distribution assay of an orally administered microalgae nanomodular integrated gel system Mice with colitis were randomly divided into four groups of 20 mice each, and fasted for 12 hours before administration. Mice were administered FKLNPs@Dio, IG@SP-FK LNPs@Dio, SP-FK LNPs@Dio, and IG@FK LNPs@Dio via gavage, respectively, at a dosage of 0.2 mg / kg of total lipids from FK LNPs. At 2 h, 6 h, 12 h, and 24 h, five mice from each group were sacrificed by cervical dislocation. The gastrointestinal tract, heart, liver, spleen, lungs, and kidneys were collected and placed in a small animal in vivo imaging system to observe the distribution of lipid nanoparticles in the isolated tissues.

[0053] like Figure 8 The fluorescence signals of FK LNPs@Dio were mainly distributed in the gastrointestinal tract. No fluorescence signals were observed in the heart, liver, spleen, lungs, or kidneys, indicating that FK LNPs@Dio was not absorbed into the bloodstream. Gastrointestinal distribution results showed that at 2 h, the fluorescence signals of FK LNPs@Dio, SP-FK LNPs@Dio, IG@SP-FK LNPs@Dio, and IG@FK LNPs@Dio were mainly distributed in the small intestine. At 6 h, the fluorescence signals of the four formulations gradually shifted towards the large intestine. At 12 h, the fluorescence signals of FK LNPs@Dio and SP-FK LNPs@Dio were very weak, and approached zero at 24 h. Compared with the control group, IG@SP-FK LNPs@Dio still showed strong fluorescence signal intensity in the cecum and colon at 12 h and 24 h.

[0054] As Figure 9 shown in FIG. 24, IG@SP-FK LNPs@Dio still has a strong fluorescence signal at the 24th hour. The above results show that inulin coating protects the lipid nanoparticles from damage in the gastrointestinal tract, and the addition of SP is conducive to the colonization of the lipid nanoparticles in the intestine.

[0055] Example 7: Tissue distribution test of oral microalgae inner nanomodular integrated gel system Healthy C57BL / 6 mice were randomly divided into 7 groups (n=10), namely normal control group, model control group, 5-ASA, IG-SP group, FK LNPs@siTNFα group, SP-FK LNPs@siTNFα group, and IG@SP-FK LNPs@siTNFα group. Except for the normal control group which was free to drink water, the rest were free to drink 3% DSS solution for 7 days to establish a mouse colitis model. During the modeling period, the mice were free to eat, and the fresh DSS solution was replaced every day to prevent deterioration. The 5-aminosalicylic acid group (5-ASA) mice were orally administered 5-ASA (150 mg / kg / d), and the rest of the administration groups were administered according to the dose of TNF-α siRNA (3.2 μg / piece / d). The normal control group and the model group mice were simultaneously administered with the same volume of normal saline. The body weight was measured every day during the experiment, and the living conditions and fecal characteristics were observed. The colon tissues of the mice were collected on the 7th day.

[0056] As Figure 10 C shows that the body weight of the normal control group of mice as a whole showed an upward trend. The body weight of the mice administered with the modeling agent slightly fluctuated in the first three days, and there was no obvious change trend. From the fourth day, the body weight of the modeling mice began to decrease, and the body weight of the model control group of mice decreased the most. The mice in the administration groups improved the body weight loss of the mice to varying degrees. Among them, IG@SP-FK LNPs@siTNFα improved the most obvious condition; 5-ASA and SP-FK LNPs@siTNFα groups had relatively obvious improvement; however, IG-SP did not significantly improve the degree of body weight loss in mice, indicating that the improvement of IG@SP-FK LNPs@siTNFα on colitis was mainly the result of the treatment of FK LNPs@siTNFα.

[0057] As Figure 10 C shows that the DAI score of the mice, IG@SP-FK LNPs@siTNFα has a significant improvement compared with the DSS model group; the rest of the groups, 5-ASA, SP-FK LNPs@siTNFα and IG-SP mice, are relatively close in terms of disease index, and also have slight improvement compared with the DSS model group.

[0058] As Figure 10 A and Figure 10As shown in Figure E, observation of the colon's appearance and measurement of the length from the lower end of the ileocecal junction to the anus, along with photographic results of the mouse intestinal segments, revealed that the colon length in the model group mice was significantly shorter than that in the normal group (p<0.01). Grossly, no formed feces were visible in the intestinal lumen; the lumen was thinner, shorter, and less elastic, and contained dark red, watery bloody stools. After drug administration, the colon shortening and bloody stools were alleviated to varying degrees in each treatment group. The IG@SP-FK LNPs group showed a significant difference, with the mouse intestinal segment length approaching that of normal mice, and formed feces visible in the intestinal lumen.

[0059] like Figure 11 A- Figure 11 As shown in Figure D, IG@SP-FK LNPs@siTNFα reduced the concentration of pro-inflammatory cytokines in the serum of colitis mice and upregulated the expression level of TGF-β1. Although the other treatment groups also alleviated the expression of inflammatory factors to varying degrees, they were significantly different from IG@SP-FK LNPs@siTNFα.

[0060] like Figure 11 As shown in Figure E, compared with the normal group, the MPO activity in the serum of the model control group mice was significantly increased, while the drug-treated groups reduced the MPO activity in the serum of IBD mice to varying degrees; compared with other drug-treated groups, IG@SP-FK LNPs@siTNFα significantly reduced the MPO level in the serum of mice.

[0061] like Figure 11 As shown in Figure F, DSS-induced colitis significantly increased intestinal permeability in mice, leading to elevated serum FITC-Dextran concentrations. Each treatment group alleviated the increased intestinal permeability caused by colitis to varying degrees. Compared to other treatment groups, the IG@SP-FK LNPs@siTNFα treatment group showed significantly lower serum FITC-Dextran levels, with concentrations similar to those in normal mice. This indicates that IG@SP-FK LNPs@siTNFα treatment prevented the increase in intestinal permeability caused by colitis, thus avoiding systemic infection.

[0062] like Figure 12 As shown, ZO-1 is a red fluorescent signal. The results show that the red fluorescent signal of ZO-1 in the colon tissue of normal control mice is significantly stronger than that in the model control group. Oral administration of IG@SP-FK@siTNFα increases the expression level of ZO-1 in the colon tissue of DSS-induced colitis mice, thus improving the downregulation of tight junctions in the intestine of colitis mice.

[0063] like Figure 13As shown, compared with the normal group of mice, the model control group showed the disappearance of crypts and goblet cells. Pathological sections of the 5-ASA, IG-SP, SP-FK LNPs @siTNFα, and FK LNPs @TNF control groups showed varying degrees of crypt loss, goblet cell loss, and inflammatory cell infiltration. Sections of the IG@SP-FK LNPs@siTNFα treatment group showed crypts and goblet cells, but no obvious inflammatory cell infiltration.

[0064] like Figure 14 As shown, the serum levels of AST, ALT, BUN, and CREA in both the treatment group and the normal group were within the normal range, demonstrating that no significant liver or kidney toxicity was observed in any of the treatment groups during the treatment period.

[0065] Example 8: Effects of oral administration of a microalgae-based nanomodular integrated gel system on the gut microbiota of mice like Figure 15 As shown, Alpha Diversity was used to analyze the diversity of the microbial community within a sample. Single-sample diversity analysis (α-diversity) reflects the richness and diversity of the microbial community within the sample. Observed species and Chao1 are richness indices, while Shannon and Simpson are diversity indices. Higher Observed species and Chao1 values ​​indicate a richer community of species; a higher Shannon index and a lower Simpson index indicate high intra-group diversity. The results of α-diversity of the gut microbiota in colitis mice are presented. Observed species and Chao1 values ​​show that, compared with the model group and the control group, the gut microbiota richness of IG@SP-FK LNPs@siTNFα mice was significantly increased; the Shannon and Simpson indices indicate no significant difference in gut microbiota diversity among the groups. These results indicate that IG@SP-FKLNPs@siTNFα can improve the richness of the mouse gut microbiota but has no significant effect on its diversity.

[0066] like Figure 16 As shown, PCA (Principal Component Analysis) is a technique for simplifying data analysis. By analyzing the composition of different sample OTUs (97% similarity), it can reflect the differences and distances between samples. PCA uses variance decomposition to reflect the differences between multiple sets of data on a two-dimensional coordinate graph, with the axes taking the two characteristic values ​​that best reflect the variance. The more similar the sample composition, the closer the distances will be reflected in the PCA graph. Figure 16It can be seen that the distance between IG@SP-FK LNPs@siTNFα group and normal group is closer, the distance between control group FK LNPs@siTNFα and model group is closer, and the intestinal flora of IG@SP-FK LNPs@siTNFα group and normal group mice has higher similarity.

[0067] As shown in Figure 17 The intestinal flora of mice at the door level is mainly composed of Bacteroidota, Firmicute, Proteobacteria and the like. Among them, the sum of the relative abundance of Bacteroidota and Firmicute in the sample is more than 80%, and in DSS-induced colitis mice, the ratio of Firmicute / Bacteroidota increases. Compared with FK LNPs@siTNFα treatment group, IG@SP-FK LNPs@siTNFα reduces the ratio of Firmicute / Bacteroidota in the intestinal flora of mice and increases the relative abundance of beneficial bacteria. The above results show that the oral microalgae inner nano-modular integrated gel system has good in vitro gene silencing ability, is stable in the upper digestive tract and colonizes in the colon. It has sufficient biological safety, can relieve the symptoms of colitis mice, improve the intestinal flora of colitis mice, and has good effect on DSS-induced colitis.

Claims

1. A cathepsin B-reduction dual-responsive biodegradable cationic lipid, characterized in that: Disulfide bonds are used to bridge the carbon chain of a fatty acid containing a cathepsin B-sensitive dipeptide fragment to obtain the product via an amide reaction.

2. The dual-responsive biodegradable cationic lipid according to claim 1, characterized in that: The lipid consists of a disulfide bond as a linker, a symmetrical cationic dipeptide fragment phenylalanine (F)-lysine (K) recognized by cathepsin B as the lipid head, and two symmetrical myristic acid (MA) moieties as the lipid tail, abbreviated as MA-FK-SS.

3. The dual-responsive biodegradable cationic lipid according to claim 1, characterized in that: The structural formula of the cathepsin B-reduction dual-responsive biodegradable cationic lipid is: 。 4. The method for preparing the dual-responsive biodegradable cationic lipid according to any one of claims 1 to 3, characterized in that: The preparation method includes reacting a lysine-phenylalanine dipeptide fragment with myristic acid to generate an intermediate product FK-MA; then reacting the intermediate product with 2,2'-dithiodiethylamine to generate the final product MA-FK-SS.

5. A biodegradable lipid nanoparticle, characterized in that: The lipid nanoparticles comprise any one of the dual-responsive biodegradable cationic lipids according to claims 1 to 3 or the dual-responsive biodegradable cationic lipids prepared by the preparation method according to claim 4, and siRNA.

6. An oral microalgae nanomodular integrated gel system, characterized in that: Spirulina and the lipid nanoparticles of claim 5 are used to obtain microalgal nanomodules through electrostatic interaction; then, a microalgal nanomodular integrated gel system is obtained by heating and cooling.

7. The application of the dual-responsive biodegradable cationic lipid of any one of claims 1 to 3, or the dual-responsive biodegradable cationic lipid prepared by the preparation method of claim 4, or the biodegradable lipid nanoparticles of claim 5, or the oral microalgae nanomodular integrated gel system of claim 6, in the preparation of nucleic acid drug delivery carriers.

8. The application according to claim 7, characterized in that: The nucleic acid drug delivery vector is used for oral administration.

9. The application of the dual-responsive biodegradable cationic lipid of any one of claims 1 to 3, or the dual-responsive biodegradable cationic lipid prepared by the preparation method of claim 4, or the biodegradable lipid nanoparticles of claim 5, or the oral microalgae nanomodular integrated gel system of claim 6, in the preparation of a drug for treating intestinal diseases.

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