A composite hydrogel targeting macrophage mitochondria, its preparation method and application

Abstract

This invention relates to the field of nanomedicine delivery systems, and more particularly to a composite hydrogel targeting macrophage mitochondria, its preparation method, and its applications. This invention prepares a ROS-responsive, injectable liposome composite hydrogel targeting macrophage mitochondria by combining cationic liposomes with a hydrogel. This hydrogel can serve as a nanomedicine delivery system suitable for local oral administration, exhibiting good biocompatibility and the ability to target mitochondria. The composite hydrogel is easy to prepare and administer, has high viscosity, and possesses characteristics such as a sustained drug release mode, minimum dose frequency, and low drug toxicity, making it advantageous for widespread application in the treatment of periodontitis-related diseases.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine delivery systems, and more particularly to a composite hydrogel that targets macrophage mitochondria, its preparation method, and its application. Background Technology

[0002] Periodontitis (PD) is a condition caused by an imbalance between bacteria and the autoimmune system, resulting in damage to the soft and hard tissues of the teeth. Mechanical removal of bacteria from the tooth surface is the preferred treatment, but with the continuous iteration and innovation of nanomedicine delivery carriers, adjunctive methods for periodontitis have fundamentally changed the approach to periodontal treatment. Choosing appropriate drug delivery routes for localized treatment of periodontitis has become crucial. Studies have reported the use of fibers, gels, strips, films, microparticles, nanoparticles, and low-dose antibacterial agents as local drug delivery systems for this disease, aiming to deliver antibacterial agents to the subgingival lesion site.

[0003] Ideal periodontal topical drug delivery systems must be easy to administer, release drugs in a controlled manner, maintain drug concentrations for extended periods, be biodegradable, have good biocompatibility, and not cause any tissue irritation. While liposomes have been used in clinical trials in the medical field, clinical evidence investigating their efficacy in periodontal disease is scarce. Furthermore, despite the wide range of advantages of nanoliposomes, several drawbacks remain: lack of targeting and specificity, no sustained drug release, instability with sudden release, and occasional oxidation and hydrolysis-like reactions of phospholipids; high cost also hinders large-scale production, making their adoption in clinical practice difficult. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a composite hydrogel that targets macrophage mitochondria, is ROS-responsive, can target macrophage mitochondria, and is suitable for local oral administration.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a method for preparing a composite hydrogel targeting macrophage mitochondria, comprising the following steps:

[0007] (1) Mix 4-carboxybenzaldehyde, EDC and DMAP and dissolve them in an organic solvent to obtain a mixed solution; under nitrogen, add 4arm-PEG-OH to the mixed solution, stir, wash and dry to obtain 4arm-PEG-CHO, add PBS to obtain 4arm-PEG-CHO solution;

[0008] (2) Add water to polylysine and stir until homogeneous; then add EDC and NHS to adjust the pH; add dopamine hydrochloride in nitrogen, react in the dark, dialyze, and freeze dry to obtain EPL-DA;

[0009] (3) DOTAP, lecithin, cholesterol, DSPE-Se-Se-PEG-NH2, DSPE-PEG-TPP, and DSPE-PEG-HA were dissolved in an organic solvent and cationic liposomes were prepared by thin film dispersion. PBS was added to obtain a cationic liposome solution.

[0010] (4) Dissolve EPL-DA in cationic liposome solution, vortex and sonicate to obtain EPL-DA solution; mix EPL-DA solution with alkali, add 4arm-PEG-CHO solution and mix, vortex and let stand to obtain the composite hydrogel.

[0011] This invention introduces dopamine modification on polylysine, thereby increasing the adhesion and antioxidant properties of the hydrogel material; under weakly alkaline conditions, the hydrogel forms gels through Schiff base bonds. This invention is based on the engineering design of liposomes: (1) After macrophages are stimulated by LPS concentration, the expression of CD44 increases in a concentration-dependent manner. This invention designs to add hyaluronic acid (HA) component. Hyaluronic acid is one of the four major receptors of CD44 and can effectively bind to M1 cells with high expression of CD44 protein on their surface; (2) Add TPP group: TPP group is a classic mitochondrial targeting structure. After the liposome is endocytosed by macrophages, the liposome can target mitochondria for drug delivery under the influence of the group; (3) Add ROS-responsive SE-SE structure, which can intelligently identify the mitochondrial oxidative stress state. In the case of excessive ROS production, it triggers the breakage of SE-SE bond to release the drug. The monomer is encapsulated in the liposome. When the composite nanomaterials reach the mitochondria, they can prevent the sudden release of drugs and control the release of drugs; (4) The inner membrane of the mitochondria is negatively charged, and cationic liposomes can rely on physical attraction to stay in the inner membrane of the mitochondria for a long time and release drugs slowly. Liposomes enter the body and are metabolized quickly. The liposomes prepared in this invention, cationic liposomes help to release drugs slowly, prolong the residence time of liposomes at local sites, minimize off-target effects, and ensure therapeutic effects. This invention prepares a ROS-responsive liposome composite injectable hydrogel that targets the mitochondria of macrophages by combining cationic liposomes with hydrogel. It can be used as a nano-drug delivery system suitable for local oral administration, has good biocompatibility, and can target mitochondria. This composite hydrogel is easy to prepare, easy to administer, has high viscosity, and has the characteristics of continuous drug release mode, minimum dose frequency and low drug toxicity, which is conducive to its widespread application in the treatment of periodontitis-related diseases.

[0012] Preferably, the organic solvent is dichloromethane.

[0013] Preferably, in step (3), the mass ratio of DOTAP, lecithin, cholesterol, DSPE-Se-Se-PEG-NH2, DSPE-PEG-TPP, and DSPE-PEG-HA is DOTAP:lecithin:cholesterol:DSPE-Se-Se-PEG-NH2:DSPE-PEG-TPP:DSPE-PEG-HA = 6.5:2:1:2.5:2.5:2.5.

[0014] Preferably, in step (4), the mass ratio of 4arm-PEG-CHO and EPL-DA is 4arm-PEG-CHO:EPL-DA = 0.2:(0.1-0.2).

[0015] When the content of EPL-DA is 10%-20%, the prepared hydrogel has good viscosity.

[0016] More preferably, the mass ratio of the mixture of 4arm-PEG-CHO and EPL-DA is 4arm-PEG-CHO:EPL-DA = 0.2:0.15.

[0017] Preferably, in step (4), the concentration of EPL-DA in the EPL-DA solution is 0.1-0.2 g / mL.

[0018] More preferably, the concentration of EPL-DA in the EPL-DA solution is 0.15 g / mL.

[0019] Preferably, in step (3), the volume ratio of cationic liposomes to PBS is cationic liposomes:PBS = 1:(10-50). At this point, the stability of the solution is optimal, and the experimental results are also better.

[0020] Preferably, the specific preparation method of the composite hydrogel includes:

[0021] 1. Synthesis of 4arm-PEG-CHO via esterification reaction

[0022] (1) Weigh 600 mg of 4-carboxybenzaldehyde, 766.8 mg of EDC and 244.32 mg of DMAP, and dissolve them in 80 mL of dichloromethane to obtain a mixed solution;

[0023] (2) Under nitrogen atmosphere, 2g of 4arm-PEG-OH was added to the mixed solution and stirred at 40℃ for 24h;

[0024] (3) Wash the organic layer (dichloromethane) three times with 80 mL of 1 M hydrochloric acid, three times with 80 mL of saturated NaHCO3, and three times with 80 mL of brine. Dry the organic layer (dichloromethane) under reduced pressure and then dry it under vacuum to obtain a white solid, which is 4arm-PEG-CHO.

[0025] 2. Synthesis of dopamine-modified polylysine (EPL-DA)

[0026] (1) Weigh 1g of polylysine (EPL) and add it to 100mL of deionized water, and stir well;

[0027] (2) Add 1.1g of EDC and 0.66g of NHS to adjust the pH to 5.0;

[0028] (3) Add 1g of dopamine hydrochloride to nitrogen atmosphere and react at room temperature in the dark for 1 day. After the reaction is complete, dialyze the product through a 1000Da dialysis bag for 3 days and freeze-dry the product to obtain dopamine-modified polylysine (EPL-DA).

[0029] 3. Preparation of cationic liposomes (Lip)

[0030] (1) Accurately weigh 6.5mg DOTAP, 2mg lecithin, 1mg cholesterol, 2.5mg DSPE-Se-Se-PEG-NH2, 2.5mg DSPE-PEG-TPP, and 2.5mg DSPE-PEG-HA and place them in a 1L round-bottom flask. Add 40mL of dichloromethane and sonicate to form a homogeneous suspension.

[0031] (2) Place the ring-bottom flask in a rotary evaporator, keep it in a constant temperature water bath at 40℃, and rotate at 150r / min. Remove the organic solvent under reduced pressure to form a uniform lipid film on the flask wall. Hydrate with 6mL of phosphate buffer (concentration of 0.33mg / mL) at 37℃ for 2h, and sonicate with an ice bath probe for 2min (35%, on for 3s, off for 1s). After sonication, cationic liposomes are obtained.

[0032] 4. Synthesis of composite hydrogels

[0033] (1) Dissolve 0.2g of 4arm-PEG-CHO in 1mL of sterile PBS, vortex for 20s, and sonicate for 1-5 minutes to form a clear 4arm-PEG-CHO solution; dissolve 10μL of cationic liposomes in 990μL of PBS to obtain a cationic liposome solution.

[0034] (2) Dissolve 0.1-0.2g of EPL-DA in 1mL of cationic liposome solution, vortex for 20s, and sonicate for 1-5 minutes until completely dissolved to obtain EPL-DA solution;

[0035] (3) Mix 150-200 μL of EPL-DA solution with 20 μL of 1M NaOH, shake for about 10 seconds, then add 150-200 μL of 4arm-PEG-CHO solution, vortex for 2-3 minutes, and let stand for 1-3 minutes to form the composite hydrogel.

[0036] Secondly, the present invention provides a composite hydrogel targeting macrophage mitochondria prepared by the above-described preparation method.

[0037] Preferably, the composite hydrogel targeting macrophage mitochondria further includes an active drug loaded in cationic liposomes.

[0038] Preferably, the active pharmaceutical ingredient includes an antibacterial drug.

[0039] Thirdly, the present invention provides the application of the above-mentioned composite hydrogel targeting macrophage mitochondria in the preparation of drugs or formulations for treating periodontitis.

[0040] The composite hydrogel prepared by this invention can target and deliver antibacterial agents to the subgingival lesion site, enabling controlled drug release and facilitating localized treatment of periodontitis.

[0041] Preferably, the drug or preparation acts on the subgingival lesion tissue.

[0042] Fourthly, the present invention provides a medicament or preparation for treating periodontitis, comprising the aforementioned composite hydrogel targeting macrophage mitochondria.

[0043] Preferably, the dosage form of the preparation includes an injection.

[0044] The beneficial effects of this invention are as follows:

[0045] This invention introduces dopamine modification onto polylysine, thereby increasing the adhesion and antioxidant properties of the hydrogel material. The liposomes are engineered with the addition of HA, TPP, and SE-SE groups, enabling them to target macrophage mitochondria for targeted drug delivery, exhibit ROS responsiveness, and controllable drug release. The cationic liposomes facilitate slow drug release, prolonging the liposome's residence time at local sites, minimizing off-target effects, and ensuring therapeutic efficacy.

[0046] This invention synthesizes cationic liposomes with hydrogels to prepare a ROS-responsive injectable hydrogel that targets macrophage mitochondria. This hydrogel can serve as a nanomedicine delivery system suitable for local oral administration, exhibiting good biocompatibility and the ability to target mitochondria. The composite hydrogel is easy to prepare and administer, has high viscosity, and possesses characteristics such as sustained drug release, minimum dose frequency, and low drug toxicity, making it advantageous for widespread application in the treatment of periodontitis-related diseases. Attached Figure Description

[0047] Figure 1 For 4arm-PEG-CHO, 4arm-PEG-OH 1 H nuclear magnetic resonance spectroscopy.

[0048] Figure 2 For EPL-DA, EPL, DA 1 H nuclear magnetic resonance spectroscopy.

[0049] Figure 3 The infrared spectra of 4arm-PEG-CHO and 4arm-PEG-OH are shown.

[0050] Figure 4 The infrared spectra of EPL-DA, EPL, and DA are shown.

[0051] Figure 5 Transmission electron microscopy images of Lip and Lip@Ecdysone.

[0052] Figure 6 The particle size distribution of Lip and Lip@Ecdysone liposomes is shown.

[0053] Figure 7 The potential diagrams are for Lip and Lip@Ecdysone.

[0054] Figure 8 This is a graph showing the results of a liposome-targeted mitochondrial assay.

[0055] Figure 9 These are images of the hydrogel before and after gelation.

[0056] Figure 10 This is a microstructure (SEM) image of the hydrogel.

[0057] Figure 11 The swelling curves are for hydrogels of different concentrations.

[0058] Figure 12 The graph shows the relationship between G' and G” of the hydrogel material and time.

[0059] Figure 13 The graph shows the relationship between G' and G” of the hydrogel material and frequency.

[0060] Figure 14 This is the force-displacement relationship curve of the hydrogel.

[0061] Figure 15 The results show the blood compatibility test results for the composite hydrogel (the red line represents the hemolysis safety threshold).

[0062] Figure 16 The graph shows the degradation curves of the composite hydrogel under enzyme-containing or enzyme-free incubation conditions.

[0063] Figure 17 This is the standard curve for α-ecdysone.

[0064] Figure 18 This is an in vitro drug release curve of the composite hydrogel incubated in PBS or 0.5 mM H2O2.

[0065] Figure 19 Transmission electron microscopy image of Lip@Ecdysone incubated with hydrogen peroxide.

[0066] Figure 20 The results are the biocompatibility test results for the composite hydrogel.

[0067] Figure 21 These are the results of in vivo imaging tests on small animals.

[0068] Figure 22 The results are from a transmission electron microscope of rat gingival tissue.

[0069] Figure 23 HE staining results of various organs of rats after injection and subsequent sacrifice. Detailed Implementation

[0070] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0071] Preparation of 1M NaOH solution: Weigh 4.00g of sodium hydroxide solid and add 100mL of water and mix well.

[0072] The sources of the experimental reagents and instruments used in this invention are shown in Tables 1 and 2:

[0073] Table 1

[0074]

[0075]

[0076] Table 2

[0077]

[0078] Example 1:

[0079] An embodiment of the composite hydrogel targeting macrophage mitochondria of the present invention; the preparation method of the composite hydrogel includes the following steps:

[0080] 1. Synthesis of 4arm-PEG-CHO via esterification reaction

[0081] (1) Weigh 600 mg of 4-carboxybenzaldehyde, 766.8 mg of EDC and 244.32 mg of DMAP, and dissolve them in 80 mL of dichloromethane to obtain a mixed solution;

[0082] (2) Under nitrogen atmosphere, 2g of 4arm-PEG-OH was added to the mixed solution and stirred at 40℃ for 24h;

[0083] (3) Wash the organic layer (dichloromethane) three times with 80 mL of 1 M hydrochloric acid, three times with 80 mL of saturated NaHCO3, and three times with 80 mL of brine. Dry the organic layer (dichloromethane) under reduced pressure and then dry it under vacuum to obtain a white solid, which is 4arm-PEG-CHO.

[0084] 2. Synthesis of dopamine-modified polylysine (EPL-DA)

[0085] (1) Weigh 1g of polylysine (EPL) and add it to 100mL of deionized water, and stir well;

[0086] (2) Add 1.1g of EDC and 0.66g of NHS to adjust the pH to 5.0;

[0087] (3) Add 1g of dopamine hydrochloride to nitrogen atmosphere and react at room temperature in the dark for 1 day. After the reaction is complete, dialyze the product through a 1000Da dialysis bag for 3 days and freeze-dry the product to obtain dopamine-modified polylysine (EPL-DA).

[0088] 3. Preparation of cationic liposomes (Lip)

[0089] (1) Accurately weigh 6.5mg DOTAP, 2mg lecithin, 1mg cholesterol, 2.5mg DSPE-Se-Se-PEG-NH2, 2.5mg DSPE-PEG-TPP, and 2.5mg DSPE-PEG-HA and place them in a 1L round-bottom flask. Add 40mL of dichloromethane and sonicate to form a homogeneous suspension.

[0090] (2) Place the ring-bottom flask in a rotary evaporator, keep it in a constant temperature water bath at 40℃, and rotate at 150r / min. Remove the organic solvent under reduced pressure to form a uniform lipid film on the flask wall. Hydrate with 6mL of phosphate buffer (concentration of 0.33mg / mL) at 37℃ for 2h, and sonicate with an ice bath probe for 2min (power 35%, cycle mode: 3 seconds on, 1 second off) until uniformly dispersed to form cationic liposomes.

[0091] 4. Synthesis of composite hydrogels

[0092] (1) Dissolve 0.2g of 4arm-PEG-CHO in 1mL of sterile PBS, vortex for 20s, and sonicate for 1-5 minutes to form a clear 4arm-PEG-CHO solution; dissolve 10μL of cationic liposomes in 990μL of PBS to obtain a cationic liposome solution.

[0093] (2) Dissolve 0.1g of EPL-DA in 1mL of cationic liposome solution (i.e., 10% EPL-DA), vortex for 20s, and sonicate for 1-5 minutes until completely dissolved to obtain EPL-DA solution.

[0094] (3) Mix 150-200 μL of EPL-DA solution with 20-40 μL of 1M NaOH (the viscosity of the hydrogel can be adjusted by adding alkali), shake for about 10 seconds, then add 150-200 μL of 4arm-PEG-CHO solution and mix, vortex for 2-3 minutes, let stand for 1-3 minutes, and the composite hydrogel can be obtained.

[0095] Example 2:

[0096] An embodiment of the composite hydrogel targeting macrophage mitochondria described in this invention.

[0097] The difference between the preparation method of the composite hydrogel and that of Example 1 is that in step 4, step (2), 0.15g of EPL-DA is dissolved in 1mL of cationic liposome solution (i.e., 15% EPL-DA), and the parameters of the remaining steps are the same as those in Example 1.

[0098] Example 3:

[0099] An embodiment of the composite hydrogel targeting macrophage mitochondria described in this invention.

[0100] The difference between the preparation method of the composite hydrogel and that of Example 1 is that in step (2) of step 4, 0.2g of EPL-DA is dissolved in 1mL of cationic liposome solution (i.e., 20% EPL-DA), and the parameters of the remaining steps are the same as those in Example 1.

[0101] Example 4:

[0102] An embodiment of the composite hydrogel targeting macrophage mitochondria described in this invention; the composite hydrogel described in this embodiment is a drug-loaded composite hydrogel.

[0103] The composite hydrogel targeting macrophage mitochondria includes an active drug (e.g., ecdysone) loaded in cationic liposomes.

[0104] The difference between the preparation method of the composite hydrogel described in this embodiment and that in Example 1 is that in step (2) of step 3, 6 mL of phosphate buffer is replaced with an equal amount of PBS solution containing Ecdysone (Ecdysone concentration is 0.33 mg / mL), and the parameters of the remaining steps are the same as those in Example 1.

[0105] Test Example 1: Characterization of Synthetic Products

[0106] Test samples: 4arm-PEG-CHO and EPL-DA prepared in Example 1; raw materials for preparation: 4arm-PEG-OH, EPL, and DA.

[0107] 1. Nuclear magnetic resonance (NMR) analysis: 15 mg of sample was weighed and dissolved in an appropriate amount of deuterated reagent (deuterated water D2O or deuterated chloroform CDCl3), then placed in a clean NMR tube, and the NMR structure was determined by an NMR spectrometer at room temperature. The spectrum was then analyzed using MestReNova software.

[0108] from 1 The 1H NMR spectroscopy results show that 4arm-PEG-CHO contains peaks of aldehyde (10.04 ppm), benzene ring (8.23 ppm, 7.99 ppm), and methylene (4.54 ppm) protons. Figure 1 This invention synthesizes EPL-DA by covalently modifying the amino group of DA onto the carboxyl group of EPL. The NMR spectrum (…) Figure 2 As can be seen, the multiplets in the 6.8 ppm–7.2 ppm region are generated by the CH protons on the dopamine benzene ring; the new peak at 2.65 ppm is attributed to the proton residues of DA; and the proton peaks in the 1.2 ppm–1.9 ppm region are generated by the -CH3 protons attached to the amide group on hyaluronic acid. These results indicate that the DA residues are effectively modified on the polylysine (EPL) backbone.

[0109] 2. Infrared Spectroscopy Measurement: First, take 3-5 mg of sample and an appropriate amount of dry potassium bromide powder (mass ratio approximately 5%) in an agate mortar and grind them thoroughly to ensure uniform mixing. Then, take an appropriate amount of the ground sample powder and compress it into a pellet (vacuum pressure 20 mmHg, compression time 5 min) to obtain a sample thin film. Set the scanning range to 4000-500 cm⁻¹. -1 The detection was performed using a Fourier transform infrared spectrometer.

[0110] Infrared spectral results are as follows Figure 3-4 As shown, 1695 cm⁻¹ in 4arm-PEG-CHO -1 The peak was assigned as the stretching vibration of the CO bond. Figure 3 This further demonstrates the successful introduction of aldehyde groups into 4arm-PEG-CHO. DA and EPL-DA were respectively measured at 1614 cm⁻¹. -1 and 1552cm -1 There are unique peaks ( Figure 4 This is due to the stretching vibration of the amide bond. These results indicate that the DA group was successfully modified onto the EPL molecule.

[0111] Test Example 2: Liposome Characterization

[0112] Samples: empty cationic liposomes Lip, and liposomes Lip@Ecdysone loaded with Ecdysone (Example 4).

[0113] 1. Transmission electron microscopy observation: The sample solution was diluted to a certain concentration, ultrasonically dispersed, and a 20 μL pipette was used to drop the solution onto a copper grid. Then, 2% phosphotungstic acid was used for negative staining, and the solution was dropped onto the copper grid. After natural evaporation, the internal tissue morphology of the liposomes was observed using a transmission electron microscope.

[0114] Transmission electron microscopy results as follows Figure 5 As shown, the liposomes prepared by this invention exhibit a spherical shape and a monodisperse distribution. The particle size of the liposomes ranges from approximately 180 nm and is uniformly distributed; while the size of the liposomes loaded with Ecdysone is slightly larger than that of the empty liposomes.

[0115] 2. DLS test: The sample solution was diluted to 100 μg / mL, ultrasonically dispersed, and the average particle size of the liposomes was tested using a Malvern particle size analyzer. At the same time, the surface charge of the liposome particles was detected.

[0116] DLS analysis revealed that the average particle sizes of Lip and Lip@Ecdysone were 189.1 nm and 195.4 nm, respectively. Figure 6 The PDI values ​​for Lip and Lip@Ecdysone were 0.123 and 0.223, respectively. The Zeta potential results are as follows: Figure 7As shown, the zeta potential of the blank Lip liposome was 36.9 mV, while the zeta potential of the liposome after binding with Ecdysone was 30.2 mV, indicating a decrease in potential.

[0117] 3. Targeted testing

[0118] Experimental steps:

[0119] (1) THP-1 cells were stimulated with PMA (phorbol ester) for 24 hours to differentiate into macrophages. The differentiated THP-1 macrophages were seeded at an appropriate density in a confocal culture dish.

[0120] (2) Dilute FITC fluorescent liposomes to an appropriate concentration and add them to THP-1 macrophages. Co-culture them in a 37°C, 5% CO2 incubator for 12 hours.

[0121] (3) After co-culture, gently wash the cells 2-3 times with preheated PBS; add diluted MitoTrackerDeepRedFM (mitochondrial dye) to a final concentration of about 100-500 nM, incubate at 37°C for 15-30 minutes, and then gently wash the cells 2-3 times with PBS again; fix the cells with 4% paraformaldehyde at room temperature for 15 minutes, and wash them twice with PBS; add DAPI solution (1 μg / mL) and stain at room temperature for 5 minutes; finally wash the cells twice with PBS.

[0122] (4) Use a confocal laser scanning microscope to observe the sample.

[0123] (5) Image analysis: Open the obtained images using ImageJ software; perform colocalization analysis using the "Coloc 2" plugin; select FITC channel (liposomes) and MitoTracker channel (mitochondria) for analysis; record Pearson's correlation coefficient (PCC) and Manders' overlap coefficient (MOC).

[0124] (6) Data analysis: Calculate the average PCC of multiple images; the PCC value ranges from -1 to 1; where 1 represents a perfect positive correlation, 0 represents no correlation, and -1 represents a perfect negative correlation. The higher the PCC, the better the targeting.

[0125] The results are as follows Figure 8 As shown. The liposomes prepared in this invention have a PCC value of 0.92 and exhibit excellent macrophage mitochondrial targeting.

[0126] Test Example 3: Hydrogel Characterization

[0127] Characterization samples: Composite hydrogels prepared in Examples 1-3.

[0128] Note: Scanning electron microscopy (SEM) observation of hydrogels requires rapid lyophilization in liquid nitrogen before imaging, as liposomes collapse. Therefore, this test case uses transmission electron microscopy (TEM) to characterize liposomes, and SEM is used in conjunction with the hydrogel (without adding cationic liposome solution during preparation, and instead using an equal amount of PBS) for observation.

[0129] 1. Appearance Characterization: The appearance of the hydrogel prepared in this invention before and after gelation is as follows: Figure 9 As shown, a 20% 4arm-PEG-CHO and 15% EPL-DA solution can form a hydrogel within 2 minutes at room temperature.

[0130] 2. Observation of the microstructure of the hydrogel using scanning electron microscopy (SEM)

[0131] The prepared 400 μL hydrogel was pre-frozen in a -20℃ freezer. After freeze-drying, gold was sputtered onto the surface of the hydrogel for 30 seconds, and the surface morphology of the hydrogel was observed using a scanning electron microscope.

[0132] The results are as follows Figure 10 As shown, the hydrogel exhibits a 3D, relatively homogeneous, and interconnected pore structure, indicating good structural stability and chemical homogeneity. The pore size of the hydrogel is significantly dependent on the EPL-DA concentration; the higher the EPL-DA concentration, the smaller the pore size. This highly porous hydrogel allows for the release of drugs during slow diffusion. Furthermore, this porous, interconnected structure provides ample space for cell growth, attachment, proliferation, and extracellular matrix secretion.

[0133] 3. Determination of hydrogel swelling rate

[0134] Weigh the initial weight (W0) of the hydrogel, and then immerse it in PBS buffer (pH = 7.4) at 37°C. Remove the hydrogel at different time points, wipe off the surface moisture with filter paper, and weigh the hydrogel (Wt). Calculate the swelling ratio according to equation (1):

[0135] Swelling rate = (Wt - W0) / W0 × 100% — Equation (1)

[0136] The results are as follows Figure 11 As shown, the hydrogels all reached swelling equilibrium after 5 hours, with maximum swelling rates of 34.4%, 21.7%, and 15.7%, respectively. The swelling properties and volume growth rate of the hydrogels are beneficial for accelerating the tissue healing process by absorbing tissue exudate, promoting hemostasis, and promoting tissue integration.

[0137] 4. Rheological properties of hydrogels

[0138] After demolding 800 μL of hydrogel, rheological measurements were performed using a 25 mm diameter stainless steel parallel plate rotor. The storage modulus (G') and loss modulus (G”) variation curves were recorded using dynamic strain frequency scanning (0.1–100 Hz, strain 1%) and time scanning (5 rad / s, strain 1%, 20 min).

[0139] To further verify the stability of the hydrogel, rheological tests were performed in this embodiment. The changes in the storage modulus (G') and loss modulus (G”) of the gel over time were measured using a rheometer at different times and angular frequencies, and the rheological properties of PEG-15% EPL-DA were obtained.

[0140] The curves showing the changes in storage modulus (G') and loss modulus (G”) of hydrogel materials over time are shown in the figure below. Figure 12 As shown, neither G' nor G” changed significantly with time, and G' was always greater than G”, indicating that the hydrogel material has good stability.

[0141] The curves showing the changes in storage modulus (G') and loss modulus (G”) of hydrogel materials with angular frequency are shown below. Figure 13 As shown, after the hydrogel forms a gel, G' is always greater than G" as the angular frequency increases, indicating that the hydrogel can exist stably in the form of a gel under suitable conditions.

[0142] 5. Adhesion test:

[0143] 200 μL of hydrogel was injected between two pieces of porcine skin tissue, each 30 mm long and 10 mm wide. An additional 200 g weight was added to the sample for 5 min to enhance adhesion. The sample was then tested at a constant tensile speed of 1 mm / min until complete separation.

[0144] Drug-loaded hydrogels typically need to adhere to tissues. For example... Figure 14 As shown, the results indicate that the peel strength of PEG-15% EPL-DA hydrogel is significantly higher than that of other hydrogel groups. This suggests that PEG-15% EPL-DA hydrogel, as an adhesive hydrogel, exhibits strong adhesion properties to porcine skin tissue. Among the three hydrogels with different proportions, PEG-15% EPL-DA showed the best adhesive strength. Furthermore, the viscosity decreased with increasing EPL-DA mass fraction. This is mainly due to the gradual increase in the number of amino groups and the binding of more aldehyde groups in EPL-DA, resulting in a decrease in the number of aldehyde groups that bind to the tissue.

[0145] Test Example 4: Performance Testing of Composite Hydrogels

[0146] Sample: Composite hydrogel prepared in Example 2.

[0147] 1. Blood compatibility test

[0148] Blood samples were collected from the hearts of healthy SD rats. After washing the anticoagulated whole blood multiple times and centrifuging (2000 rpm, 5 min), red blood cells were precipitated. The red blood cells were mixed with phosphate-buffered saline (PBS) at a volume ratio of 1:16 to obtain a solution rich in red blood cells (RBCs), which was used for subsequent testing of the hemolytic properties of the hydrogel.

[0149] One mL of solidified hydrogel sample was immersed in a mixture of 1 mL RBC solution and 4 mL PBS, and incubated at 37°C for 4 h and 8 h. The solution was then centrifuged at 3500 r / min for 5 min, and the absorbance of the supernatant at 540 nm was measured using an ELISA reader. The hemolysis rate (H) was calculated according to formula (2). Positive and negative control groups were also set up. The positive control group was a mixture of 1 mL RBC and 4 mL deionized water (blood was hemolyzed in water), and the negative control group was a mixture of 1 mL RBC solution and 4 mL PBS.

[0150] Hemolysis rate (H)% = (Dt-Dnc) / (Dpc-Dnc)×100% — Equation (2)

[0151] In the above formula: Dt, Dnc, and Dpc are the absorbance of the sample, negative control group, and positive control group, respectively.

[0152] The hemolysis test is a common method for evaluating the destructive effect of blood red blood cells on red blood cells when they come into contact with a material. The better the blood compatibility of the material, the lower the hemolysis rate. The hemolysis test results of the hydrogel sample prepared in this invention are as follows: Figure 15 As shown, the hemolysis rates of both hydrogel samples were lower than the industry standard of 5%, indicating that the hydrogels have good blood compatibility.

[0153] 2. In vitro degradation performance test

[0154] First, freeze-dry the hydrogel for testing and accurately weigh it as W0. Then, immerse other test samples in PBS solution containing 0 or 1000 U / mL lysozyme and place them in a constant temperature shaker (37℃, 70 rpm). At the measurement time point, wash the hydrogel with ultrapure water, freeze-dry it, and weigh it as Wt. Calculate the weight retention rate of the hydrogel according to formula (3) using the following formula:

[0155] Weight retention rate (%) = Wt / W0 × 100% — Equation (3)

[0156] The biodegradability of the hydrogel was evaluated by studying its degradation in PBS buffer solution and lysozyme / PBS buffer solution. The weight change curves of the hydrogel after immersion in PBS buffer solution and 1000 U / mL lysozyme / PBS buffer solution for a certain period of time are shown below. Figure 16 As shown, after 6 days of immersion in PBS solution, the hydrogel's weight retention rate was 39.3%, mainly due to degradation caused by the hydrolysis of ester bonds within the hydrogel. However, in PBS buffer solution containing lysozyme, the hydrogel completely degraded by day 8, indicating good biodegradability. The degradation rate of the hydrogel is relatively suitable for the tissue healing process. In the early stages of tissue healing, the hydrogel can maintain stability and antibacterial effects; then, the hydrogel enters a disintegration phase and gradually undergoes complete biodegradation, which will simplify subsequent hydrogel removal.

[0157] 3. In vitro drug release test

[0158] (1) Detect encapsulation efficiency and drug loading rate

[0159] A stock solution of α-ecdysone with a concentration of 1 mg / mL was precisely prepared and then serially diluted to standard solutions of 125, 62.5, 31.25, 15.625, 7.8125, and 3.90625 μg / mL.

[0160] The selected liquid chromatography conditions were acetic acid:water:methanol = 60:40:0.1, flow rate 1 mL / min, injection volume 20 μL, and detection wavelength 242 nm. A standard curve equation was obtained by plotting concentration on the x-axis and peak area on the y-axis and performing linear regression. The prepared standard solution was stored at 4 °C.

[0161] The encapsulation efficiency and drug loading of liposomes in the composite hydrogel were determined by ultrafiltration centrifugation. 1 mL of the prepared sample solution was accurately measured and placed in an ultrafiltration centrifuge tube (with a molecular weight cutoff of 4000). The tube was centrifuged at 8000 r / min for 20 min at 4℃. 200 μL of the solution obtained after centrifugation of the outer tube was taken, and 200 μL of methanol was added. The concentration of α-ecdysone in the mixed solution was determined using a UV spectrophotometer. The ecdysone content was calculated based on the standard curve, thus obtaining the amount of free ecdysone. The encapsulation efficiency and drug loading of liposomes in the composite hydrogel were calculated using the following formulas (4) and (5):

[0162] Encapsulation efficiency (%) = (Amount of ecdysin during drug loading - Content of free ecdysin) / Amount of melatonin during drug loading × 100% — Formula (4)

[0163] Drug loading rate (%) = Ecdysin content in liposomes / Total mass of liposomes × 100% — Formula (5)

[0164] The ecdysone standard curve obtained in this embodiment is as follows: Figure 17 As shown, the encapsulation efficiency of liposomes in the composite hydrogel was 96.5%; the drug loading rate was 14.5%.

[0165] (2) Place the composite hydrogel in 5 mL of PBS or 0.5 mM hydrogen peroxide in a PBS solution and incubate it in a constant temperature shaker (37℃, 70 rpm). Collect the PBS supernatant at different time points and replace it with the same volume of PBS solution. Determine the concentration of α-ecdysin in the collected supernatant by high performance liquid chromatography based on the peak area value of the pre-established standard curve, and calculate the cumulative release rate according to formula (6):

[0166] Cumulative release rate (%) = Cumulative drug release / Total drug content × 100% — Formula (6)

[0167] The microenvironment of inflamed tissues in the human body is a high-ROS environment. By investigating the release levels of drugs under different environments, we can understand the characteristics of drug carriers and their effects on human tissues. Results are as follows... Figure 18 As shown, the release rate of α-ecdysone in the hydrogel was significantly higher under hydrogen peroxide conditions than in the PBS environment, suggesting that reactive oxygen species triggered liposome cleavage under H2O2 treatment.

[0168] To demonstrate liposome fragmentation triggered by reactive oxygen species under H2O2 treatment, Lip@Ecdysone was observed by TEM after 5 minutes of incubation in H2O2. TEM images show ( Figure 19 The spherical structure of Lip@Ecdysone was disrupted after incubation with H2O2, indicating that H2O2 caused the breakage of the diselenide bonds in the liposome components, leading to the rupture of Lip@Ecdysone. Therefore, in the presence of hydrogen peroxide, the liposomes in the composite hydrogel rupture, resulting in a relatively faster release of α-ecdysone; demonstrating that the composite hydrogel prepared in this invention is ROS-responsive.

[0169] 4. Biocompatibility testing

[0170] THP-1 cells were induced with PMA for 24 hours, and after adhesion, they became macrophages. At a concentration of 5 × 10⁶ cells / year... 3 Cells were seeded per well in 96-well plates, and a composite hydrogel sample was added to the cells. A control group (without composite hydrogel) was set up. Cells were incubated at 37°C in a 5% CO2 incubator for 24, 72, and 120 h, respectively. At each time point, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for another 4 h. The absorbance was measured at 450 nm using a microplate reader.

[0171] The results are as follows Figure 20As shown. The composite hydrogel exhibits good biocompatibility when co-cultured with macrophages, with toxicity levels of 0–1.

[0172] 5. Small animal in vivo imaging experiment

[0173] A periodontitis model rat was selected (the periodontitis model was established by ligating the maxillary second molar with nylon wire for 1 week and then removing the sutures). 40 μL of FITC-labeled sample was injected into the gingival tissue. The rats were observed using an IVIS in vivo imaging system at 0, 1, 2, 3, 4, 5, 6 and 7 days.

[0174] The results are as follows Figure 21 As shown, the fluorescence signal of the composite hydrogel sample is mainly concentrated at the injection site and can be maintained for 7 days; the signal gradually weakens but is always detectable, indicating that the composite hydrogel sample prepared in this invention has good local retention and stability, and is suitable for the treatment needs of periodontitis.

[0175] 6. In vivo validation of targeting mitochondria in inflammatory macrophages of gingival tissue

[0176] A rat model of periodontitis was selected, and 40 μL of FITC-labeled sample was injected into the gingival tissue. Seven days later, the gingival tissue was collected for sample fixation and ultrathin sectioning. The distribution of the composite hydrogel sample in macrophages was observed using transmission electron microscopy (TEM).

[0177] The results are as follows Figure 22 As shown, the sample (marked with a yellow arrow) was localized within the mitochondria (marked with a red arrow) of macrophages. Rough endoplasmic reticulum (marked with a blue arrow) and lysosomes (marked with a white arrow) were also observed, indicating that the composite hydrogel sample successfully targeted the macrophage mitochondria of gingival tissue. This result further validates the precise targeting and delivery capability of the composite hydrogel and its effectiveness at sites of inflammation.

[0178] 7. In vivo safety evaluation

[0179] 40 μL of the sample was injected into the gingival tissue of rats with periodontitis. The drug was administered every other day for 4 consecutive weeks. After that, the major organs (heart, liver, spleen, lung, and kidney) of the rats were collected, fixed, sectioned, and stained with hematoxylin and eosin (HE) to observe the toxicity and pathological changes of the composite material on the major organs. A normal group and a periodontitis model group (lig was the modeling group) were set up as controls.

[0180] The results are as follows Figure 23 As shown in the figure, no obvious inflammation, necrosis, or other pathological changes were observed in the tissue sections of the heart, liver, spleen, lungs, and kidneys of rats in each group. The structures of the major organs were intact and similar to those of the normal group, and no toxic effects were observed. The results indicate that the composite hydrogel prepared in this invention has good biocompatibility and in vivo safety.

[0181] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a composite hydrogel targeting macrophage mitochondria, characterized in that, Includes the following steps: (1) Mix 4-carboxybenzaldehyde, EDC and DMAP and dissolve them in an organic solvent to obtain a mixed solution; under nitrogen, add 4arm-PEG-OH to the mixed solution, stir, wash and dry to obtain 4arm-PEG-CHO, add PBS to obtain 4arm-PEG-CHO solution; (2) Add water to polylysine and stir until homogeneous; then add EDC and NHS to adjust the pH; add dopamine hydrochloride in nitrogen, react in the dark, dialyze, and freeze dry to obtain EPL-DA; (3) DOTAP, lecithin, cholesterol, DSPE-Se-Se-PEG-NH2, DSPE-PEG-TPP, and DSPE-PEG-HA were dissolved in an organic solvent and cationic liposomes were prepared by thin film dispersion. PBS was added to obtain a cationic liposome solution. (4) Dissolve EPL-DA in cationic liposome solution, vortex and sonicate to obtain EPL-DA solution; mix EPL-DA solution with alkali, add 4arm-PEG-CHO solution and mix, vortex and let stand to obtain the composite hydrogel.

2. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of DOTAP, lecithin, cholesterol, DSPE-Se-Se-PEG-NH2, DSPE-PEG-TPP, and DSPE-PEG-HA is DOTAP:lecithin:cholesterol:DSPE-Se-Se-PEG-NH2:DSPE-PEG-TPP:DSPE-PEG-HA = 6.5:2:1:2.5:2.5:2.

5.

3. The preparation method according to claim 1, characterized in that, In step (4), the mass ratio of 4arm-PEG-CHO and EPL-DA is 4arm-PEG-CHO:EPL-DA=0.2:(0.1-0.2).

4. The preparation method according to claim 1, characterized in that, In step (4), the concentration of EPL-DA in the EPL-DA solution is 0.1-0.2 g / mL.

5. The composite hydrogel targeting macrophage mitochondria prepared by the preparation method according to any one of claims 1-4.

6. The composite hydrogel targeting macrophage mitochondria as described in claim 5, characterized in that, The composite hydrogel targeting macrophage mitochondria also includes an active drug loaded in cationic liposomes.

7. The use of the composite hydrogel targeting macrophage mitochondria as described in claim 5 or 6 in the preparation of a medicament for treating periodontitis.

8. The application as described in claim 7, characterized in that, The drug acts on the subgingival lesion tissue.

9. A preparation for treating periodontitis, characterized in that, The composite hydrogel that targets macrophage mitochondria as described in claim 5 or 6.

10. The formulation as described in claim 9, characterized in that, The dosage form of the preparation includes injections.

Images