Preparation method and application of 4-octyl itaconic acid loaded liposome composite hydrogel microspheres

Abstract

The invention discloses a lipidosome composite hydrogel microsphere for repairing damaged intestinal mucosa and a preparation method thereof, LHMs (at) 4-OI is composed of hyaluronic acid modified targeted drug-loaded lipidosome, sodium alginate, hydrochloric acid treated sucralfate and a CaCl cross-linking agent, and rapid gel forming is carried out under the voltage of 11.0 KV through an electrospray technology. The microspheres have pH responsiveness and gastric juice digestion resistance, CD44 receptor targeting is achieved through hyaluronic acid modification, sodium alginate and Ca form an egg box structure to provide a three-dimensional network, and sucralfate enhances adhesion to isolate harmful substances. The preparation method comprises the steps of preparation of the drug-loaded liposome by a film hydration method, hyaluronic acid modification and electrospray crosslinking, is simple to operate and low in cost, and can be used for large-scale production. The microspheres can effectively load an anti-inflammatory drug 4-OI, inhibit expression of inflammatory factors and promote self-repair of damaged intestinal mucosa, and have wide application prospects in the fields of abdominal infection, inflammatory bowel diseases, colorectal cancer and the like.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a liposome composite hydrogel microsphere (LHMs@4-OI) loaded with 4-octylitaconic acid (4-OI) for oral and targeted drug delivery, and its preparation method. Background Technology

[0002] Open abdominal surgery is an effective treatment for severe abdominal infections. However, after opening the abdominal cavity, the abdominal organs are exposed to a non-physiological environment for an extended period. Secondary damage to the exposed intestinal segments can exacerbate intestinal immune and metabolic disorders, resulting in a mortality rate as high as 30%. The two main causes of intestinal damage after open abdominal surgery are: ① damage to the serosa caused by the exposed intestinal segments; ② damage to the mucosal barrier due to intestinal inflammatory response. Intestinal mucosal damage can lead to the invasion of large numbers of bacteria, viruses, and endotoxins, activating the immune system and inducing a cytokine storm, further aggravating the intestinal mucosal damage. Therefore, to promote the healing of damaged intestinal mucosa, prevent the invasion of harmful substances, and deliver anti-inflammatory drugs in a targeted manner, appropriate drug delivery systems are needed.

[0003] As a derivative of itaconic acid in the tricarboxylic acid cycle, 4-OI also possesses a similar electrophilic α,β-unsaturated π-π conjugated structure, enabling it to undergo Michael addition reactions with nucleophiles, affecting the activity of substrate proteins and thus exerting a certain anti-inflammatory effect. With anti-digestion and pH responsiveness, LHMs@4-OI, on the one hand, prevents the invasion of harmful substances through its own adhesion, laying the spatiotemporal foundation for drug targeting; on the other hand, by slowly releasing anti-inflammatory drugs, it precisely targets CD44, promoting intestinal barrier repair while simultaneously combating inflammation.

[0004] Chinese invention patent application CN202211402539.0 discloses a drug-loaded liposome hydrogel, its preparation, and its application. The drug-loaded liposome hydrogel is composed of a drug-loaded carboxymethyl chitosan-sodium alginate hydrogel encapsulating drug-loaded liposomes. Specifically, the drug-loaded liposomes are liposomes carrying acetylshikonin and aloe-emodin, and the drug-loaded carboxymethyl chitosan-sodium alginate hydrogel is a carboxymethyl chitosan-sodium alginate hydrogel cross-linked with asiaticoside. This drug-loaded liposome hydrogel achieves sustained release, continuous release, and sequential delivery of drugs through different drug delivery methods. It addresses the different needs of wound repair at different stages of wound recovery in terms of anti-inflammation and cell growth, forming a drug release mode corresponding to the needs, thereby shortening the healing time of burn wounds, reducing scar tissue formation, and achieving better wound repair. It provides an excellent new raw material for wound-promoting medical dressings. However, this invention lacks oral administration capability and is not suitable for the repair of intestinal mucosal damage; furthermore, the liposomes do not possess active targeting capabilities.

[0005] Chinese invention patent application CN202111188654.8 discloses a self-renewing hydrating lubricating drug-loaded hydrogel microsphere, its preparation method, and its application. This patent combines methacrylated hyaluronic acid with cationic liposomes loaded with rapamycin through non-covalent interactions. Further, through microfluidic technology and photo-initiated free radical polymerization, a monodisperse, uniformly sized lipid-hydrogel microsphere with both self-renewing lubrication and cellular homeostasis maintenance functions is obtained. This microsphere exhibits good biocompatibility, can encapsulate liposomes to form a "reservoir," and continuously exposes the encapsulated liposomes when the surface wears down, forming a self-renewing hydrating lubricating layer, while also acting as a "rolling bearing" for lubrication. The cationic liposomes released from the microspheres can target and bind to the cartilage surface to release drugs, significantly alleviating articular cartilage wear, inhibiting cartilage tissue degeneration and osteophyte formation, and delaying the progression of osteoarthritis. However, the photoinitiators used in this invention generally have potential cytotoxicity, and their main applications are bone wear and maintaining joint self-lubrication. Moreover, the yield of microfluidic technology is extremely low, which is not conducive to mass production and has little value for clinical translation. Summary of the Invention

[0006] In view of the shortcomings and deficiencies of the prior art, the present invention aims to provide a liposome composite hydrogel microsphere (LHMs@4-OI) loaded with 4-octyl itaconic acid for treating damage to the intestinal mucosal barrier.

[0007] The technical solution of the present invention is as follows:

[0008] A method for preparing liposome-based hydrogel microspheres loaded with 4-octyl itaconic acid, the method comprising the following steps:

[0009] (1) Hydrogenated soybean lecithin HSPC, cholesterol CHOL and DSPE-MPEG2000 are dissolved in a mixed solvent, and then 4-octyl itaconic acid 4-OI is added. After the addition is complete, the organic solvent is vacuum evaporated on a rotary evaporator to form a lipid membrane containing 4-OI. The lipid membrane is first evaporated at 1~5℃ for 10~14h to remove the residual solvent. After the residual solvent is evaporated, it is first hydrated and then granulated 5~15 times using a liposome extruder to obtain a lipo@4-OI solution loaded with 4-OI.

[0010] (2) Place the sodium hyaluronate solution into a dialysis bag and dialyze it in dilute hydrochloric acid solution. After dialysis, freeze-dry the desalted sodium hyaluronate. Incubate the freeze-dried sodium hyaluronate with lipo@4-OI solution at room temperature for 20-40 min, and then incubate it at 1-5℃ for 2-5 h. After granulation 5-15 times by a liposome extruder, the HA-modified HA-lipo@4-OI solution can be obtained.

[0011] (3) Sucralfate is placed in a dialysis bag with a molecular weight cutoff of 10-20 kDa and dialyzed in 0.1-0.5 mol / L dilute hydrochloric acid. Then, it is dialyzed for 2-4 h at 1-5℃ to remove other small molecule impurities. After dialysis, the liquid in the dialysis bag is taken out, its pH value is neutralized to 4-5, and after freeze drying, sucralfate colloidal powder can be obtained.

[0012] (4) Take sodium alginate and sucralfate colloidal powder obtained by (3) and prepare them into a mixed solution of sodium alginate and sucralfate HA-lipo@4-OI; draw it into a 5 ml syringe, set the voltage to 10.0~15.0 KV, the pump speed to 0.25-0.75 mm / min, and the nozzle size to 20-25G, start the electrospray instrument, and the droplets fall into the receiving container. The receiving container contains 5%-15% CaCl2 (w / v) as a crosslinking agent to promote the rapid formation of liposome composite hydrogel microspheres.

[0013] In the above preparation method, the molar ratio of hydrogenated soybean lecithin HSPC, cholesterol CHOL and DSPE-MPEG2000 in step (1) is 5~15:5~10:0.1~2; the mixed solvent is chloroform and methanol with a volume ratio of 1~3:1~3.

[0014] In the above preparation method, W in step (1) 4-OI :W 总脂 The mass ratio is 1:5~15, where W 总脂 This refers to the total mass of hydrogenated soybean lecithin (HSPC), cholesterol (CHOL), and DSPE-MPEG2000, W. 4-OI It is the quality of 4-OI.

[0015] In the above preparation method, in step (1), hydration involves adding deionized water or PBS to the 4-OI lipid membrane after evaporating the residual solvent, followed by treatment at 35-15℃ and 110-130 rpm for 5-8 h. Preferably: W 4-OI The mass-to-volume ratio of the PBS to deionized water or PBS is 25-35 mg : 8-12 mL.

[0016] In the above preparation method, the concentration of HA in the HA-lipo@4-OI solution in step (2) is 40~60% of the concentration of 4-OI.

[0017] In the above preparation method, the w / v concentration of sodium alginate in the HA-lipo@4-OI mixed solution in step (4) is 1~5%, and the w / v concentration of sucralfate colloid powder is 0.25~1%.

[0018] In the above preparation method, the model of the electrospray instrument in step (4) is BT-2135. The conditions for preparing liposome composite hydrogel microspheres are a voltage of 11.0 KV, a 22G nozzle, a pumping speed of 0.5 mm / min, and a CaCl2 solution with a w / v concentration of 8~12% as a crosslinking agent to ensure rapid crosslinking to form liposome composite hydrogel microspheres.

[0019] A liposome-based hydrogel microsphere loaded with 4-octyl itaconic acid was prepared using the method described above.

[0020] In the technical solution of this invention, the liposome composite hydrogel microspheres loaded with 4-octyl itaconic acid prepared by the method are used for self-repair and anti-inflammatory effects on damaged intestinal mucosal barrier.

[0021] In the technical solution of this invention, w / v refers to mass-volume percentage, that is, the mass of solute in grams contained in 100 mL of solution.

[0022] As described in step (4) of the preparation method above, the w / v concentration of sodium alginate in the HA-lipo@4-OI mixed solution is 1~5%, which means that 100mL of HA-lipo@4-OI mixed solution contains 1~5g of sodium alginate; the w / v concentration of sucralfate colloidal powder is 0.25~1%, which means that 100mL of HA-lipo@4-OI mixed solution contains 0.25~1g of sodium alginate.

[0023] As in step (4) above, a CaCl2 solution with a w / v concentration of 10% represents 8~12g of CaCl2 in 100mL of CaCl2 solution.

[0024] In this invention, the hyaluronic acid-modified liposomes primarily function as precise targets and drug loading agents. The core principle of the cross-linking of sodium alginate with CaCl2 is that Ca... 2+ Ionic cross-linking with the guluronic acid units on the sodium alginate molecular chain forms an "egg-box structure." The interaction between sodium alginate and the cross-linking agent CaCl2 is crucial for the preparation of LHMs@4-OI and is also key to the pH responsiveness and gastric acid resistance of the entire delivery system. Sucralfate increases the viscoelasticity of LHMs@4-OI and enables it to adhere to the damaged intestinal mucosa after reaching the site of injury, preventing the invasion of bacteria, viruses, and endotoxins in the intestinal lumen and avoiding the induction of inflammatory storms that further aggravate intestinal damage.

[0025] In the technical solution of this invention, hydrogenated soybean lecithin is the main membrane material constituting the liposomes during the preparation of cationic liposomes. It serves as an important structural support for the formation of the phospholipid bilayer and exhibits good biocompatibility and stability. Cholesterol insertion into the phospholipid bilayer can regulate membrane fluidity and stability, reduce drug leakage, enhance antioxidant capacity, and increase in vivo circulation time. DSPE-mPEG2000 is a PEGylated phospholipid that can provide a "stealth" effect for the liposomes, reduce reticuloendothelial system clearance, prolong circulation time in the blood, and can also serve as a bridge connecting target ligands (such as connecting HA). Hyaluronic acid (HA), a natural polysaccharide, boasts high biocompatibility. On one hand, it effectively neutralizes the positive charge on the surface of cationic liposomes, reducing their toxicity and immunogenicity to normal cells. Through the formation of a hydration layer on the liposome surface, HA enhances their stability in the physiological environment and enables pH-responsive release, facilitating microenvironment-triggered drug release. On the other hand, HA, through electrostatic adsorption and physical adsorption with cationic liposomes, can specifically bind to CD44 or hyaluronic acid-mediated motor receptor (CD168) overexpressed on the surface of various tumor cells or inflammatory cells, achieving active targeted drug delivery and significantly improving drug accumulation efficiency at lesion sites. The attached 4-OI, through alkylation modification of the C147 site of STING, inhibits STING activation and thereby suppresses the production of downstream inflammatory factors, exhibiting excellent anti-inflammatory effects and creating favorable conditions for the self-repair of damaged intestinal mucosa.

[0026] The LHMs@4-OI prepared by the above method can be used for self-repair and anti-inflammatory treatment of damaged intestinal mucosal barrier.

[0027] The beneficial effects of this invention are:

[0028] 1. Inspired by the structure and function of natural extracellular vesicles and the regulatory mechanisms of the extracellular matrix microenvironment, this invention constructs a liposome-based hydrogel microsphere drug delivery system. This system aims to address the problems of burst release, poor targeting, and lack of environmental responsiveness inherent in traditional delivery systems, achieving a technological breakthrough in high stability, controlled release, and targeted therapy. The system is then applied to the repair and anti-inflammatory treatment of damaged intestinal mucosa. The liposome-based hydrogel microspheres are prepared by electrospray ionization at 25°C and 11.0 KV using HA-modified liposomes loaded with 4-OI, sodium alginate, sucralfate treated with dilute hydrochloric acid, and 10% CaCl2 crosslinking agent. The microspheres are loaded with 4-OI and exhibit CD44 targeting, anti-digestion properties, and pH responsiveness. Sodium alginate not only provides a rigid three-dimensional network structure for the entire drug delivery system but also provides space for drug-loaded liposomes. The loading of sucralfate enables the entire drug delivery system to possess a certain degree of adhesion, prolonging its retention time in the intestines. This not only creates timeliness for successful liposome targeting but also forms a physical barrier, preventing harmful substances such as bacteria, viruses, and endotoxins from invading the body, activating the immune system, and causing a vicious cycle. Its particle size distribution is uniform, with an average particle size in the micrometer range, making it suitable for oral gavage administration.

[0029] 2. This invention modifies cationic liposomes through physical adsorption, successfully preparing liposomes that target CD44, overcoming the defect of 4-OI not having active targeting, enabling 4-OI to be recognized by macrophages more efficiently, thereby mediating their endocytosis, shortening the action time of 4-OI, effectively inhibiting the process of inflammation, and creating favorable conditions for the self-repair of damaged intestinal mucosa.

[0030] 3. This liposome-hydrogel microsphere delivery system combines the advantages of both liposomes and hydrogels, effectively solving the problem that most liposomes cannot be taken orally. Moreover, sodium alginate, after gelation, has certain resistance to gastric digestion and pH responsiveness, which to some extent broadens the range of drugs it can carry.

[0031] 4. Both the preparation of liposomes and the preparation of liposome-composite hydrogel microspheres are simple to operate, use readily available raw materials, are inexpensive, and can be mass-produced. Compared with microfluidic preparation of liposomes, the thin-film hydration method is simple to operate, low in cost, and has low equipment requirements; while the electrospray method for preparing composite microspheres has advantages over microfluidic preparation in that it requires simple equipment, has mild conditions, high encapsulation efficiency, and is easy to construct composite systems, making it particularly suitable for the development of biomolecule delivery and sustained-release systems.

[0032] 5. The liposome composite hydrogel microspheres of the present invention can effectively inhibit the expression of inflammatory factors in the body, have a certain adhesion ability, effectively isolate the external environment, avoid the invasion of harmful substances, and significantly promote the self-repair of damaged intestinal mucosa.

[0033] 6. The liposome-based composite hydrogel microspheres described in this invention have also shown wide applications in other regenerative medicine fields, such as drug delivery for inflammatory bowel disease, tumors (e.g., colorectal cancer), gut microbiota dysbiosis-related diseases, or neurological diseases (oral delivery of neurotrophic factors to promote nerve regeneration). Therefore, these easily prepared and stored liposome-based composite hydrogel microspheres demonstrate great potential and application value in the treatment of intestinal mucosal injury, providing a new therapeutic strategy for promoting the healing of damaged intestinal mucosa. Attached Figure Description

[0034] Figure 1 The negative staining, particle size and zeta potential diagrams of the 4-OI-loaded liposomes (lipo@4-OI) synthesized by the thin-film hydration method in Example 1 are shown.

[0035] Figure 2 Negative staining, particle size, and zeta potential of liposomes (HA-lipo@4-OI) in Example 2 and after modification with 50% HA;

[0036] Figure 3 This is a comparison chart of the polydispersity index (PDI) of lipo@4-OI and HA-lipo@4-OI in Example 3;

[0037] Figure 4 This is a flow cytometry diagram of HA-mediated macrophage endocytosis in Example 3;

[0038] Figure 5 This is an optical microscope image of the liposome composite hydrogel microspheres (LHMs@4-OI) synthesized by electrospray technology in Example 3.

[0039] Figure 6 This is a graph showing the change in particle size under different voltage conditions in Example 3;

[0040] Figure 7 The image shows the SEM image of LHMs@4-OI in Example 3;

[0041] Figure 8 The FTIR plot of LHMs@4-OI in Example 3;

[0042] Figure 9 .a is the DSC diagram of LHMs@4-OI in Example 3;

[0043] Figure 9.b is the TGA graph of LHMs@4-OI in Example 3;

[0044] Figure 10 This is a drug release diagram of LHMs@4-OI in simulated gastric and intestinal fluids in Example 3;

[0045] Figure 11 This is an adhesion diagram of LHMs@4-OI on the small intestine in Example 3;

[0046] Figure 12 The image shows the retention of LHMs@4-OI in the gastrointestinal tract of SD rats in Example 3.

[0047] Figure 13 This is a diagram showing the healing of damaged intestinal mucosa in SD rats treated with LHMs@4-OI in Example 3.

[0048] Figure 14 This is a graph showing the detection of inflammatory factors in the damaged intestinal mucosa tissue of SD rats treated with LHMs@4-OI in Example 3. Detailed Implementation

[0049] This invention provides liposome-based composite hydrogel microspheres for the treatment of damaged intestinal mucosa and a method for preparing the same. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention. All reagents used in the embodiments of this invention are commercially available.

[0050] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0051] Those skilled in the art should understand that, in the following description of the embodiments of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0052] Those skilled in the art will understand that the numerical ranges in the embodiments of this application should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0053] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While this application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this application.

[0054] Example 1: Preparation of 4-OI-loaded liposomes (lipo@4-OI)

[0055] Simply put, hydrogenated soybean lecithin: cholesterol: DSPE-mPEG2000 is dissolved in a molar ratio of 11:8:1 in a 1:1 volume mixture of chloroform and methanol, and then the drug-lipid ratio is 1:10 (W). 4-OI :W 总脂 30.8 mg of 4-OI was added to the mixture, and the organic solvent was vacuum evaporated at 40°C and 100 rpm in a rotary evaporator to form a uniform lipid film containing 4-OI. The residual solvent was allowed to evaporate overnight at 4°C. 10 mL of PBS (pH=7.4) was accurately measured and added to the round-bottom flask after the residual organic solvent had evaporated overnight at 4°C. The lipid film was then hydrated at 40°C and 120 rpm for 1 hour. After the lipid film adhering to the wall was completely hydrated, it was sonicated for 10 seconds in a water bath. After 10 granulation cycles using a liposome extruder, the resulting liposomes carrying 4-OI (lipo@4-OI) were stored at 4°C in the dark for later use. The preparation process for lipo@C6 was the same.

[0056] Example 2: Preparation of HA-modified liposomes for screening the optimal concentration

[0057] First, 50 mg of sodium hyaluronate was dissolved completely in 150 mL of deionized water and then transferred to a dialysis bag (MWCO: 8000-14000). The solution was dialyzed in dilute hydrochloric acid (0.01 M) for 8 h, and then freeze-dried to obtain hyaluronic acid (HA), which was stored at 4°C for later use. To investigate the optimal concentration of HA-modified liposomes for targeting, 4-OI was replaced with coumarin 6 (C6, concentration 1 mg / ml), a fluorescent substance with the same lipophilicity. Immunofluorescence was used to investigate the HA-mediated endocytosis of M1 macrophages by lipo@C6.

[0058] Accurately weigh lyophilized hyaluronic acid (HA) and prepare lipo@C6 solutions containing 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, and 1.00 mg / mL HA. Incubate at room temperature for 30 min, then at 4°C for 4 h. After 10 granulation cycles using a liposome extruder, solutions with concentrations of 25% HA-lipo@C6, 50% HA-lipo@C6, 75% HA-lipo@C6, and 100% HA-lipo@C6 are obtained. After multiple filtrations and granulation using a liposome extruder, the liposomes are stored in brown sample vials at 4°C protected from light. All liposomes were filtered through a 0.22 μm sterile filter before animal gavage and used within two weeks of preparation.

[0059] The preparation method of 50% HA-lipo@4-OI is the same as above. The concentration of HA in the 50% HA-lipo@4-OI solution is 50% of the concentration of 4-OI.

[0060] Example 3: Preparation of liposome-composite hydrogel microspheres

[0061] Synthesis of liposome-based hydrogel microspheres loaded with 4-OI (LHMs@4-OI).

[0062] Sucralfate was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed in 0.3 mol / L dilute hydrochloric acid. Then, it was dialyzed at 4°C for 3 h to remove other small molecule impurities. After dialysis, the liquid in the dialysis bag was taken out, its pH value was neutralized to 4.5, and after freeze-drying, sucralfate colloidal powder was obtained.

[0063] Sodium alginate (Alg) and sucralfate (Su) colloidal powder treated with hydrochloric acid (0.3 mol / L) were dissolved in a 50% HA-lipo@4-OI solution to prepare a mixed solution of HA-lipo@4-OI containing 2% (w / v) sodium alginate and 0.5% (w / v) sucralfate. The solution was drawn into a 5 ml syringe. Under the conditions of 11.0 kV voltage, 0.5 mm / min pump speed, and 22 G nozzle size, the electrospray instrument was started. The position of the receiving container was adjusted appropriately according to the droplet falling distance. The receiving container contained 10% CaCl2 (w / v) solution as a crosslinking agent to promote the rapid formation of LHMs@4-OI droplets.

[0064] Because the experiments involve animals, all solutions must be filtered with a 0.22 μm sterile filter before the experiments.

[0065] Figure 1The figures show the negative staining, particle size, and zeta potential of lipo@4-OI. As shown in the figure, (a) is the negative staining of lipo@4-OI; (b) shows the average particle size of lipo@4-OI, which is 171.51 ± 0.27 nm; and (c) shows the zeta potential of lipo@4-OI, which is -25.23 ± 0.70 mV.

[0066] Figure 2 The figures show the negative staining, particle size, and zeta potential of HA-lipo@4-OI. As shown in the figure, (a) is the negative staining of HA-lipo@4-OI; (b) shows the average particle size of HA-lipo@4-OI, which is 185.43 ± 0.29 nm; and (c) shows the zeta potential of HA-lipo@4-OI, which is -31.93 ± 0.30 mV.

[0067] Figure 3 This is a comparison chart of the polydispersity index (PDI) of lipo@4-OI and HA-lipo@4-OI. Their PDI values ​​are 0.13±0.02 and 0.227±0.05, respectively.

[0068] Figure 4 (a) is a flow cytometry graph of macrophage endocytosis mediated by different concentrations of HA. (b) is a quantitative analysis of the average positive rate at 3 h and 6 h in (a). The average positive rate analysis showed that at 3 h, the average positive rate of macrophage endocytosis mediated by 50% HA was 13.36 times that of lipo@C6, and at 6 h, the average positive rate of macrophage endocytosis mediated by 50% HA was 5.10 times that of lipo@C6. Given the significant ability of 50% HA to mediate lipoendocytosis, we finally chose the concentration of HA as 50% of the concentration of the drug carried, and labeled 50% HA-lipo as HA-lipo in subsequent studies.

[0069] Figure 5 This is an optical microscope image of liposome-based hydrogel microspheres (LHMs@4-OI) synthesized using electrospray ionization technology. When the sucralfate concentration is 0.25%, the microspheres do not form spherical shapes and have wrinkled, uneven surfaces. At a sucralfate concentration of 1%, the microspheres form spherical shapes, but their large particle size is unfavorable for later gavage administration to SD rats. However, when the sucralfate concentration is 0.5%, the entire composite microsphere surface is smooth and even with a uniform particle size distribution.

[0070] Figure 6This graph shows the change in the particle size of liposome-composite hydrogel microspheres under different voltage conditions. We investigated the changes in microsphere particle size under different voltage conditions (10.0 kV, 11.0 kV, and 12.0 kV) using 2% Alg. It can be seen that the microsphere particle size decreases as the voltage gradually increases, indicating an inverse relationship between the two. However, for experimental safety and to meet practical application requirements, we ultimately chose 11.0 kV as the voltage for our subsequent experiments.

[0071] Figure 7 This is a SEM image of LHMs@4-OI. The SEM images after freeze-drying show that it is a regular spherical or slightly collapsed spherical shape with a complete outline and no cracks. Its three-dimensional porous honeycomb skeleton lays a certain foundation for the later release of HA-lipo@4-OI.

[0072] Figure 8 This is the FTIR spectrum of LHMs@4-OI. To ensure that the functional groups of 4-OI remained unchanged during the preparation of the liposome-composite hydrogel microspheres, we used FTIR analysis to confirm that 4-OI did not lose its pharmacological activity. The results show that the stretching vibration peak of the ester carbonyl group (C=O) is located at 1726 cm⁻¹. −1 At this point, the stretching vibration peak of the carbon-carbon double bond (C=C) in olefins is located at 1635 cm⁻¹. −1 The asymmetric C–O–C stretching vibration peak appears at ~1650–1600 cm⁻¹. −1 The C–H stretching vibration peak is located at 2959 cm⁻¹. −1 2923cm −1 and 2854 cm −1 These characteristic peaks indicate that 4-OI did not undergo esterification or amidation reactions during the preparation of LHMs@4-OI. However, at 1738 cm⁻¹... −1 and 1097 cm −1 The shift in the absorption peak indicates that hydrogen bonds have formed between C=O and -OH groups, causing the peak positions of C=O and COC to shift.

[0073] Figure 9 Figure .a shows the DSC plot of LHMs@4-OI. To investigate whether the composite microspheres affect the storage conditions after 4-OI loading, we used DSC to investigate the thermodynamic properties of the composite microspheres. DSC data showed that the initial melting point of the liposome-based hydrogel microspheres loaded with 4-OI was 48.3℃, the peak temperature was 66.0℃, the final melting temperature was 75.1℃, and the enthalpy of melting was only 14.27 J / g. The extremely low enthalpy and wide melting range (26.8℃) indicate that the composite system is mainly amorphous with only a small amount of effective crystalline region, which is beneficial for drug sustained release, unlike 4-OI and HA-lipo@4-OI.

[0074] Figure 9 Figure .b shows the TGA image of the synthesized LHMs@4-OI. The TGA results show that the liposome-coated hydrogel microspheres carrying 4-OI experienced mass loss between 237.9℃ and 322.4℃, with a first-stage mass change of −58.47% and a second-stage mass change of −18.48%, peaking at 293.2℃ and 403.9℃ respectively, resulting in a final residual mass of 23.00%. These results indicate that the liposome-coated hydrogel microspheres carrying 4-OI exhibit better thermal stability at high temperatures than both 4-OI and HA-lipo@4-OI, suggesting their suitability for room-temperature storage, especially with the higher residual mass demonstrating better stability.

[0075] Figure 10 The figures show the drug release of LHMs@4-OI in simulated gastric and intestinal fluids as determined by HPLC. (a) shows the drug release of LHMs@4-OI in simulated gastric fluid. Free 4-OI in simulated gastric fluid is rapidly released within 1 h. Although the release rate of the HA-lipo@4-OI group is not fast, its drug release is still higher than that of the liposome-coated hydrogel microsphere group, which swells when bound to the composite microspheres in simulated gastric fluid. This indicates that the liposome-coated hydrogel microspheres have a certain degree of anti-swelling ability in simulated gastric fluid, thereby reducing the loss of 4-OI when reaching the site of intestinal injury. (b) shows the drug release of LHMs@4-OI in simulated intestinal fluid. The drug release rate of all groups in simulated intestinal fluid is faster than that in simulated gastric fluid. This indicates that water absorption and hydrogel swelling in simulated intestinal fluid lead to pore enlargement and accelerate drug release. In summary, the liposome-based hydrogel microspheres carrying 4-OI effectively resisted gastric acid erosion, ensuring that most of the drug could reach the target tissue, providing a solid foundation for the successful targeting of HA-lipo@4-OI. Furthermore, due to the properties of sodium alginate itself and the form in which the carboxylic acid groups exist, in an acidic environment, the carboxylic acid groups exist in the –COOH (protonated) form, resulting in enhanced hydrogen bonds between molecular chains and increased interchain interactions, leading to chain contraction, reduced solubility, and prevention of gel swelling. In a slightly alkaline environment, the carboxylic acid groups deprotonate, becoming –COO... - It exists in the form of (carboxylate ions), which are negatively charged. The molecular chains extend due to electrostatic repulsion, increasing the water absorption and swelling of the gel.

[0076] Figure 11 This was to verify the adhesion of sucralfate to liposome-based hydrogel microspheres on the pig small intestine. Compared with the microspheres without sucralfate, the sliding distance of the mixture was significantly shortened after the addition of sucralfate, indicating that sucralfate increased the adhesiveness of the mixture to some extent. (b) is a quantitative analysis of the sliding distance of the mixture containing carbon black b-labeled microspheres in (a).

[0077] Figure 12 This study validated the retention of sucralfate-containing liposome-based hydrogel microspheres in the gastrointestinal tract of SD rats. DIR was used as a fluorescent marker, and small animal imaging was employed to observe changes in fluorescence signals in each group. Results showed that the fluorescence intensity of the microspheres with added sucralfate was significantly enhanced and lasted longer in the gastrointestinal tract, indicating that it effectively prolonged the retention time of the microspheres in this region. This property helps improve the drug release efficiency at the target site, further supporting the feasibility of in vivo application of HA-lipo@4-OI.

[0078] Figure 13 The following are images showing the healing of damaged intestinal mucosa in SD rats treated with LHMs@4-OI: (a) is a representative HE staining image of newly formed tissue of damaged intestinal mucosa, and (b) is the quantitative statistics of (a).

[0079] Figure 14 This image shows the detection of inflammatory factors in the damaged intestinal mucosa of SD rats treated with LHMs@4-OI. The results showed that the expression levels of inflammatory factors such as TNF-α, IL-6, IL-1β, and IFN-β were significantly reduced in the intestinal tissue of rats treated with liposome-based hydrogel microspheres loaded with 4-OI (LHMs@4-OI), indicating that this microsphere system has a good anti-inflammatory effect. Simultaneously, compared with the untreated group, the treated group showed significantly greater intestinal mucosal healing, with intact and well-arranged newly formed tissue structures, demonstrating a high tissue repair capacity. These results fully demonstrate that liposome-based hydrogel microspheres loaded with 4-OI can effectively alleviate intestinal inflammation and promote mucosal repair, representing a promising therapeutic strategy for intestinal diseases.

Claims

1. A method for preparing liposome-based hydrogel microspheres loaded with 4-octyl itaconic acid, characterized in that, The method includes the following steps: (1) Hydrogenated soybean lecithin HSPC, cholesterol CHOL and DSPE-MPEG2000 are dissolved in a mixed solvent, and then 4-octyl itaconic acid 4-OI is added. After the addition is complete, the organic solvent is vacuum evaporated on a rotary evaporator to form a lipid membrane containing 4-OI. The lipid membrane is first evaporated at 1~5℃ for 10~14h to remove the residual solvent. After the residual solvent is evaporated, it is first hydrated and then granulated 5~15 times using a liposome extruder to obtain a lipo@4-OI solution loaded with 4-OI. (2) Place the sodium hyaluronate solution into a dialysis bag and dialyze it in dilute hydrochloric acid solution. After dialysis, freeze-dry the desalted sodium hyaluronate. Incubate the freeze-dried sodium hyaluronate with lipo@4-OI solution at room temperature for 20-40 min, and then incubate it at 1-5℃ for 2-5 h. After granulation 5-15 times by a liposome extruder, the HA-modified HA-lipo@4-OI solution can be obtained. (3) Sucralfate is placed in a dialysis bag with a molecular weight cutoff of 10-20 kDa and dialyzed in 0.1-0.5 mol / L dilute hydrochloric acid. Then, it is dialyzed for 2-4 h at 1-5℃ to remove other small molecule impurities. After dialysis, the liquid in the dialysis bag is taken out, its pH value is neutralized to 4-5, and after freeze drying, sucralfate colloidal powder can be obtained. (4) Take sodium alginate and sucralfate colloidal powder obtained by (3) and prepare them into a mixed solution of sodium alginate and sucralfate HA-lipo@4-OI; draw it into a 5 ml syringe, set the voltage to 10.0~15.0 KV, the pump speed to 0.25-0.75 mm / min, and the nozzle size to 20-25G, start the electrospray instrument, and the droplets fall into the receiving container. The receiving container contains 5%-15% CaCl2 (w / v) as a crosslinking agent to promote the rapid formation of liposome composite hydrogel microspheres.

2. The method for preparing liposome-based composite hydrogel microspheres loaded with 4-octyl itaconic acid according to claim 1, characterized in that, In step (1), the molar ratio of hydrogenated soybean lecithin HSPC, cholesterol CHOL and DSPE-MPEG2000 is 5~15:5~10:0.1~2; the mixed solvent is chloroform and methanol in a volume ratio of 1~3:1~3.

3. The method for preparing liposome-based hydrogel microspheres loaded with 4-octyl itaconic acid according to claim 1, characterized in that, In step (1), W 4-OI :W 总脂 The mass ratio is 1:5~15, where W 总脂 This refers to the total mass of hydrogenated soybean lecithin (HSPC), cholesterol (CHOL), and DSPE-MPEG2000, W. 4-OI It is the quality of 4-OI.

4. The method for preparing liposome-based hydrogel microspheres loaded with 4-octyl itaconic acid according to claim 1, characterized in that, In step (1), hydration involves adding deionized water or PBS to the 4-OI lipid membrane after the residual solvent has evaporated, and then treating it at 35-15℃ and 110-130 rpm for 5-8 h.

5. The method for preparing liposome-based hydrogel microspheres loaded with 4-octyl itaconic acid according to claim 1, characterized in that, In step (2), the concentration of HA in the HA-lipo@4-OI solution is 40-60% of the concentration of 4-OI.

6. The method for preparing liposome-based hydrogel microspheres loaded with 4-octyl itaconic acid according to claim 1, characterized in that, In step (4), the w / v concentration of sodium alginate in the HA-lipo@4-OI mixed solution is 1~5%, and the w / v concentration of sucralfate colloid powder is 0.25~1%.

7. The method for preparing liposome-based hydrogel microspheres loaded with 4-octyl itaconic acid according to claim 1, characterized in that, In step (4), the model of the electrospray instrument is BT-2135. The conditions for preparing liposome composite hydrogel microspheres are: voltage of 11.0 KV, nozzle of 22G, pump speed of 0.5 mm / min, and CaCl2 solution with a w / v concentration of 8~12% as a crosslinking agent to ensure rapid crosslinking to form liposome composite hydrogel microspheres.

8. A liposome-based hydrogel microsphere loaded with 4-octyl itaconic acid, characterized in that, It is prepared by the method described in any one of claims 1 to 7.

9. The application of the liposome-loaded hydrogel microspheres of 4-octyl itaconic acid prepared by the method of claim 1 in the self-repair and anti-inflammatory effects of damaged intestinal mucosal barrier.

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