Composite hydrogel targeting macrophage mitochondria as well as preparation method and application of composite hydrogel

Abstract

The invention relates to the field of nano drug delivery systems, in particular to composite hydrogel targeting macrophage mitochondria as well as a preparation method and application of the composite hydrogel. The ROS response type liposome composite injectable hydrogel targeting the macrophage mitochondria is prepared by compounding the cationic liposome and the hydrogel, can be used as a nano drug delivery system suitable for oral local drug delivery, has good biocompatibility, and can target the mitochondria. The composite hydrogel is easy to prepare, easy to administrate and high in viscosity, has the characteristics of continuous drug release mode, minimum dosage frequency, low drug toxicity and the like, and is beneficial to being widely applied to treatment of periodontitis related diseases.

Description

Technical Field

[0001] The present invention relates to the field of nano-drug delivery systems, and particularly to a composite hydrogel targeting macrophage mitochondria, its preparation method and application. Background Art

[0002] Periodontitis (PD) is a defect of the periodontal soft and hard tissues caused by the imbalance between bacterial microorganisms and the body's immune system. Mechanical removal of bacteria on the tooth surface is the preferred treatment for periodontitis. However, with the continuous iteration and update of nano-drug delivery carriers, the adjuvant methods for periodontitis have completely changed the ways and means of periodontal treatment. Selecting an appropriate drug delivery route to carry drugs can locally regulate and treat the periodontium, and has become the key to periodontal treatment. Research has reported that fibers, gels, strips, films, microparticles, nanoparticles and low-dose antibacterial agents are local drug delivery systems available for this disease, aiming to deliver antibacterial agents to the subgingival lesion sites.

[0003] Ideal local drug delivery for the periodontium must be easy to administer, release drugs in a controllable manner, maintain drug concentration for a long time, and be biodegradable, have good biocompatibility, and not cause any irritation to tissues. Although liposomes have been used in clinical trials in the medical field, in the aspect of periodontal diseases, there is little clinical evidence to investigate their efficacy. In addition, despite the extensive advantages of nano-liposomes, there are still some disadvantages: such as lack of targeting, specificity, no drug sustained release, and instability, sudden release may occur, and sometimes phospholipids will undergo oxidation and hydrolysis-like reactions; high cost, affecting large-scale production, making it difficult to enter clinical practice. Summary of the Invention

[0004] An object of the present invention is to overcome the deficiencies of the prior art and provide a composite hydrogel targeting macrophage mitochondria, which is ROS-responsive, can target macrophage mitochondria, and is suitable for local oral drug delivery.

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

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

[0007] (1) Mix 4-carboxybenzaldehyde, EDC and DMAP, 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, and add PBS to obtain a 4arm-PEG-CHO solution;

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

[0009] (3) Dissolve DOTAP, lecithin, cholesterol, DSPE-Se-Se-PEG-NH 2 , DSPE-PEG-TPP, and DSPE-PEG-HA in an organic solvent, and prepare cationic liposomes by the thin film dispersion method, and add PBS to obtain a cationic liposome solution;

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

[0011] In the present invention, dopamine modification is introduced onto polylysine, thereby increasing the adhesion and antioxidant properties of the hydrogel material; in a weakly alkaline environment, the hydrogel forms a gel through Schiff base bonds. The present invention conducts an engineered design based on liposomes: (1) After macrophages are stimulated by the LPS concentration, the expression of CD44 increases in a concentration-dependent manner. The present invention designs to add hyaluronic acid (HA) component, and hyaluronic acid is one of the four major receptors of CD44, which can effectively bind to M1 cells with high surface expression of CD44 protein; (2) Add TPP group: The TPP group is a classic targeting mitochondrial structure. After the liposome is phagocytosed by macrophages, under the influence of the group, the liposome can target mitochondria for drug delivery; (3) Add a ROS-responsive group SE-SE structure, which can intelligently identify the mitochondrial oxidative stress state. In the case of excessive production of ROS, trigger the cleavage of the SE-SE bond to release the drug. Encapsulate the monomer in the liposome. When the composite nanomaterial reaches the mitochondria, it can avoid the sudden release of the drug and release the drug controllably; (4) The inner mitochondrial membrane is negatively charged, and cationic liposomes can rely on physical attraction to stay in the inner mitochondrial membrane for a long time and release the drug slowly. Liposomes enter the systemic circulation and metabolize relatively quickly. For the liposomes prepared in the present invention, cationic liposomes help to release the drug slowly, extend the residence time of the liposomes at the local site, minimize the off-target effect, and ensure the therapeutic effect. The present invention combines cationic liposomes with hydrogels to prepare a ROS-responsive liposome composite injectable hydrogel targeting macrophage mitochondria, which can be used as a nanodrug delivery system suitable for local oral drug delivery, has good biocompatibility, and can target mitochondria. The composite hydrogel is easy to prepare, easy to administer, has high viscosity, has characteristics such as a sustained drug release mode, a minimum dose frequency, and low drug toxicity, and is conducive to wide application in the treatment of periodontitis-related diseases.

[0012] Preferably, the organic solvent is dichloromethane.

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

[0014] Preferably, in the step (4), the mass ratio of 4arm-PEG-CHO and EPL-DA in the mixture 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 4arm-PEG-CHO and EPL-DA in the mixture is 4arm-PEG-CHO: EPL-DA = 0.2: 0.15.

[0017] Preferably, in the 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 the step (3), the volume ratio of the cationic liposome to PBS is cationic liposome: PBS = 1: (10 - 50). At this time, the stability of the solution is the best, and the experimental effect is also more excellent.

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

[0021] 1. Synthesize 4arm-PEG-CHO through an 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, add 2 g of 4arm-PEG-OH to the mixed solution and stir at 40 °C for 24 h;

[0024] (3) Wash three times with 80 mL of 1 M hydrochloric acid, wash three times with 80 mL of saturated NaHCO 3 wash three times, wash three times with 80 mL of brine; Partially dry the organic layer (dichloromethane) under reduced pressure, and after drying under vacuum, a white solid is obtained, which is 4arm-PEG-CHO.

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

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

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

[0028] (3) In nitrogen, add 1 g of dopamine hydrochloride, and react at room temperature in the dark for 1 day. After the reaction is completed, dialyze with a 1000 Da dialysis bag for three days, and lyophilize the product to obtain dopamine-modified polylysine (EPL-DA).

[0029] 3. Preparation of cationic liposomes Lip

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

[0031] (2) Place the round-bottom flask in a rotary evaporator, keep the water bath at 40 °C, the rotation speed is 150 r / min, and evaporate the organic solvent under reduced pressure to form a uniform lipid-like film on the bottle wall; Hydrate with 6 mL of phosphate buffer solution (concentration 0.33 mg / mL) at 37 °C for 2 h, and ultrasonicate with an ice bath probe for 2 min (35%, on for 3 s, off for 1 s). After ultrasonication, cationic liposomes are obtained.

[0032] 4. Synthesis of composite hydrogel

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

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

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

[0036] In a second aspect, the present invention provides a composite hydrogel targeting macrophage mitochondria prepared by the above preparation method.

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

[0038] Preferably, the active drug comprises an antibacterial drug.

[0039] In a third aspect, the present invention provides the use of the above composite hydrogel targeting macrophage mitochondria in the preparation of a drug or preparation for treating periodontitis.

[0040] The composite hydrogel prepared by the present invention can target and deliver an antibacterial agent to the subgingival lesion site, with controlled drug release, which is beneficial for the local regulation and treatment of periodontitis.

[0041] Preferably, the site of action of the drug or preparation is the subgingival lesion tissue.

[0042] In a fourth aspect, the present invention provides a drug or preparation for treating periodontitis, comprising the above composite hydrogel targeting macrophage mitochondria.

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

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

[0045] In the present invention, dopamine modification is introduced onto polylysine, thereby increasing the adhesion and antioxidant properties of the hydrogel material; an engineered design is carried out on the liposome, adding HA components and TPP, SE-SE groups, making the liposome have macrophage mitochondria targeting ability, capable of targeted drug delivery, having ROS responsiveness, controllable drug release, and the cationic liposome helps slow down drug release, prolonging the residence time of the liposome at the local site and minimizing the off-target effect to ensure the treatment effect.

[0046] The present invention prepares a ROS-responsive liposome composite injectable hydrogel targeting macrophage mitochondria by compounding cationic liposomes and hydrogels, which can be used as a nano-drug delivery system suitable for oral local drug delivery, has good biocompatibility, and can target mitochondria. This composite hydrogel is easy to prepare, easy to administer, has high viscosity, has characteristics such as a sustained drug release pattern, a minimum dose frequency, and low drug toxicity, and is conducive to wide application in the treatment of periodontitis-related diseases. Description of the Drawings

[0047] Figure 1 1H nuclear magnetic resonance spectra of 4arm-PEG-CHO and 4arm-PEG-OH 1

[0048] Figure 2 1H nuclear magnetic resonance spectra of EPL-DA, EPL, and DA 1

[0049] Figure 3 Infrared spectra of 4arm-PEG-CHO and 4arm-PEG-OH

[0050] Figure 4 Infrared spectra of EPL-DA, EPL, and DA

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

[0052] Figure 6 Particle size distribution diagrams of Lip and Lip@Ecdysone liposomes

[0053] Figure 7 Potential diagrams of Lip and Lip@Ecdysone

[0054] Figure 8 Test result diagrams of liposome targeting mitochondria

[0055] Figure 9 Appearance diagrams of the hydrogel before and after gelation

[0056] Figure 10 Observation diagrams of the microstructure (SEM) of the hydrogel

[0057] Figure 11 Swelling curves of hydrogels with different concentrations

[0058] Figure 12 Diagrams of the relationship between G’ and G” of the hydrogel material and time

[0059] Figure 13 Diagrams of the relationship between G’ and G” of the hydrogel material and frequency ​​

[0060] Figure 14 It is the relationship curve of the force and displacement of the hydrogel.

[0061] Figure 15 It is the blood compatibility test result of the composite hydrogel (the red line is the hemolysis safety threshold).

[0062] Figure 16 It is the degradation curve of the composite hydrogel incubated under enzymatic or non-enzymatic conditions.

[0063] Figure 17 It is the standard curve of α-ecdysone.

[0064] Figure 18 It is for the composite hydrogel incubated in PBS or 0.5 mM H 2 O 2 environment in vitro drug release curve graph.

[0065] Figure 19 It is the transmission electron microscope image of Lip@Ecdysone incubated with hydrogen peroxide.

[0066] Figure 20 It is the biocompatibility test result of the composite hydrogel.

[0067] Figure 21 It is the test result of small animal in vivo imaging.

[0068] Figure 22 It is the transmission electron microscope result of rat gingival tissue.

[0069] Figure 23 It is the HE staining result of each organ after injection and sacrifice of the rat materials in each group. Specific implementation mode

[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 4.00 g of sodium hydroxide solid and add 100 mL of water and mix evenly.

[0072] The sources of the experimental reagents and experimental instruments used in the present invention are shown in Table 1 and Table 2 as follows:

[0073] Table 1

[0074]

[0075]

[0076] Table 2

[0077]

[0078] Example 1:

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

[0080] 1. Synthesize 4arm-PEG-CHO through an 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, add 2 g of 4arm-PEG-OH to the mixed solution and stir at 40 °C for 24 h;

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

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

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

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

[0087] (3) Under nitrogen, add 1 g of dopamine hydrochloride, and react at room temperature in the dark for 1 day. After the reaction is completed, dialyze with a 1000 Da dialysis bag for three days, and lyophilize the product to obtain dopamine-modified polylysine (EPL-DA).

[0088] 3. Prepare cationic liposome Lip

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

[0090] (2) Place the round-bottom flask in a rotary evaporator, maintain a constant water bath temperature of 40 °C, with a rotation speed of 150 r / min, and evaporate the organic solvent under reduced pressure to form a uniform lipid-like thin film on the inner wall of the flask; hydrate with 6 mL of phosphate buffer solution (concentration 0.33 mg / mL) at 37 °C for 2 h, and then probe sonicate in an ice bath for 2 min (power 35%, cycle mode: on for 3 s, off for 1 s), sonicate and disperse until uniform to form cationic liposomes.

[0091] 4. Synthesis of composite hydrogel

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

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

[0094] (3) Mix 150 - 200 μL of the EPL-DA solution with 20 - 40 μL of 1 M NaOH (the viscosity of the hydrogel can be adjusted by the amount of base added), shake for about 10 s, then add 150 - 200 μL of the 4arm-PEG-CHO solution and mix, vortex for 2 - 3 min, and let stand for 1 - 3 min to form the gel and obtain the composite hydrogel.

[0095] Example 2:

[0096] An example of the composite hydrogel targeting macrophage mitochondria of the present invention.

[0097] The difference in the preparation method of the composite hydrogel from that of Example 1 is: in step (2) of step 4, dissolve 0.15 g of EPL-DA in 1 mL of the 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 example of the composite hydrogel targeting macrophage mitochondria of the present invention.

[0100] The difference in the preparation method of the composite hydrogel from that of Example 1 is: in step (2) of step 4, dissolve 0.2 g of EPL-DA in 1 mL of the 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 example of the composite hydrogel targeting macrophage mitochondria according to the present invention; the composite hydrogel in this example is a drug-loaded composite hydrogel.

[0103] The composite hydrogel targeting macrophage mitochondria includes an active drug (taking ecdysone as an example) loaded in cationic liposomes.

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

[0105] Test Example 1: Characterization of the synthesized product

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

[0107] 1. Proton nuclear magnetic resonance analysis: Weigh 15 mg of each sample and dissolve it in an appropriate amount of deuterated reagent (heavy water D 2 O or deuterated chloroform CDCl 3 ), then load it into a clean nuclear magnetic tube, and perform nuclear magnetic structure determination using a nuclear magnetic resonance spectrometer at room temperature, and use MestReNova software for spectrum analysis.

[0108] From the 1 H nuclear magnetic resonance spectrum results, it can be seen that there are peaks of aldehyde (10.04 ppm), benzene ring (8.23, 7.99 ppm), and methylene (4.54 ppm) protons in 4arm-PEG-CHO ( Figure 1 ). In the present invention, the amino group of DA is modified to the carboxyl group of EPL through a covalent bond reaction to synthesize EPL-DA. From the nuclear magnetic resonance spectrum ( Figure 2 ), it can be seen that the multiplet peaks in the region of 6.8 ppm - 7.2 ppm are generated by the C-H protons on the benzene ring of dopamine; the new peak at 2.65 ppm is attributed to the proton residue of DA; the proton peaks in the region of 1.2 ppm - 1.9 ppm are generated by the -CH 3 protons connected to the amide group on hyaluronic acid. These results indicate that the DA residue has been effectively modified on the main chain of polylysine (EPL).

[0109] 2. Infrared Spectroscopy Determination: First, take 3 - 5 mg of the sample and an appropriate amount of dry potassium bromide powder (mass ratio is about 5%) in an agate mortar, and grind them thoroughly to make them evenly mixed. Then, take an appropriate amount of the ground sample powder for pressing tablets (vacuum pressure 20 mmHg, pressing for 5 min) to obtain a sample thin film. Set the scanning range to 4000 - 500 cm -1 , and use a Fourier transform infrared spectrometer for detection.

[0110] The results of the infrared spectrogram are as Figures 3-4 shown. The peak at 1695 cm -1 in 4arm - PEG - CHO is assigned to the stretching vibration of the C - O bond ( Figure 3 ), further indicating that an aldehyde group has been successfully introduced into 4arm - PEG - CHO. DA and EPL - DA have specific peaks at 1614 cm -1 and 1552 cm -1 respectively ( Figure 4 ), which is due to the stretching vibration of the amide bond. These results indicate that the DA group has been successfully modified on the EPL molecule.

[0111] Test Example 2: Liposome Characterization

[0112] Samples: Empty cationic liposome Lip, liposome Lip@Ecdysone encapsulating Ecdysone (Example 4).

[0113] 1. Transmission Electron Microscopy Observation: Dilute the sample solution to a certain concentration, disperse it by ultrasound, use a 20 μL pipette to drop the solution onto a copper grid, and then stain it negatively with 2% phosphotungstic acid and drop it onto the copper grid. After natural evaporation to dryness, observe the internal tissue morphology of the liposome using a transmission electron microscope.

[0114] The results of the transmission electron microscopy are as Figure 5 shown. The morphology of the liposomes prepared in the present invention shows spherical shape and monodisperse distribution. The particle size range of the liposomes is about 180 nm, with uniform distribution; while the size of the liposomes loaded with Ecdysone is slightly larger than that of the empty liposomes.

[0115] 2. DLS Test: Dilute the sample solution to 100 μg / mL, disperse it by ultrasound, and then use a Malvern particle size analyzer to measure the average particle size of the liposomes and detect the surface charge of the liposome particles simultaneously.

[0116] Through the DLS test, the average particle sizes of Lip and Lip@Ecdysone are 189.1 nm and 195.4 nm respectively ( Figure 6 ), and the PDIs of Lip and Lip@Ecdysone are 0.123 and 0.223 respectively. The Zeta potential results are as Figure 7As shown, the Zeta potential of the blank Lip liposome was 36.9 mV, while after the liposome was combined with Ecdysone, the Zeta potential was 30.2 mV, showing a decrease in potential.

[0117] 3. Targeting test

[0118] Experimental procedure:

[0119] (1) Stimulate THP-1 cells with PMA (phorbol ester) for 24 hours to differentiate them into macrophages. Seed the differentiated THP-1 macrophages in a confocal special culture dish at an appropriate density.

[0120] (2) Dilute the liposome with FITC fluorescence to an appropriate concentration and add it to THP-1 macrophages, and co-culture them in an incubator at 37 °C and 5% CO 2 for 12 hours.

[0121] (3) After the co-culture, gently wash the cells 2 - 3 times with pre-warmed PBS; add the diluted MitoTracker Deep Red FM (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, wash with PBS 2 times; add DAPI solution (1 μg / mL), stain at room temperature for 5 minutes; finally wash with PBS 2 times.

[0122] (4) Observe the samples using a confocal laser scanning microscope.

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

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

[0125] The results are as Figure 8 shown. The PCC value of the liposome prepared in the present invention is 0.92, showing excellent macrophage mitochondrial targeting.

[0126] Test Example 3: Hydrogel characterization

[0127] Samples for characterization: The composite hydrogels prepared in Examples 1 - 3.

[0128] Note: For observing the hydrogel by scanning electron microscopy, it needs to be rapidly freeze-dried with liquid nitrogen before shooting, and the liposomes will collapse. Therefore, in this test example, transmission electron microscopy was used to characterize the liposomes, and scanning electron microscopy was combined to observe the hydrogel (without adding the cationic liposome solution during the preparation process, and replacing it with an equal amount of PBS).

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

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

[0131] Place 400 μL of the prepared hydrogel in a -20°C refrigerator for pre-freezing. After freeze-drying, spray gold on the surface of the hydrogel for 30 s, and use a scanning electron microscope to observe the surface morphology of the hydrogel.

[0132] The results are as Figure 10 shown. The hydrogel shows a 3D, relatively uniform and interconnected pore structure, indicating good structural stability and uniform chemical structure of the hydrogel. The pore size of the hydrogel has a significant dependence on the EPL-DA concentration. The greater the EPL-DA concentration, the smaller the pore size of the gel. This highly porous hydrogel can release drugs during a slow diffusion process. In addition, this porous interconnected structure provides sufficient space for cell growth, attachment and proliferation, as well as the secretion of the extracellular matrix.

[0133] 3. Determination of the swelling ratio of the hydrogel

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

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

[0136] The results are as Figure 11 shown. The hydrogels all reach swelling equilibrium after 5 h, and the maximum swelling ratios of the hydrogels are 34.4%, 21.7% and 15.7% respectively. The swelling properties and volume growth rate of the hydrogel are beneficial to accelerating the tissue healing process by absorbing tissue exudates, promoting hemostasis and promoting tissue integration.

[0137] 4. Rheological properties of the hydrogel

[0138] After demolding 800 μL of the hydrogel respectively, rheological measurements were carried out using a stainless-steel parallel plate rotor with a diameter of 25 mm. The dynamic strain frequency sweep (0.1 - 100 Hz, strain 1%) and time sweep (5 rad / s, strain 1%, 20 min) were used to record the change curves of the storage modulus (G') and loss modulus (G").

[0139] To further confirm the stability of the hydrogel, a rheological test was conducted in this example. The rheometer was used to measure the changes of the storage modulus (G') and loss modulus (G") of the gel over time at different times and different angular frequencies, and the rheology of PEG-15% EPL-DA was obtained.

[0140] The graph of the change of the storage modulus (G') and loss modulus (G") of the hydrogel material over time is as Figure 12 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 graph of the change of the storage modulus (G') and loss modulus (G") of the hydrogel material over the angular frequency is as Figure 13 shown. When the hydrogel formed a gel, as the angular frequency increased, G' was always greater than G", indicating that the hydrogel could stably exist in the form of a gel under suitable conditions.

[0142] 5. Adhesion test:

[0143] Inject 200 μL of the hydrogel between two porcine skin tissues that are 30 mm long and 10 mm wide. Add an external weight of 200 g (weight) to the specimen for 5 min to enhance the adhesion. Subsequently, the test was carried out at a constant tensile speed of 1 mm / min until the sample was completely separated.

[0144] The hydrogel loaded with drugs usually needs to adhere to the tissue. As Figure 14 shown, the results indicate that the peel strength of the PEG-15% EPL-DA hydrogel is significantly higher than that of other hydrogel groups. This shows that the PEG-15% EPL-DA hydrogel, as an adhesive hydrogel, exhibits strong adhesion performance to porcine skin tissue. Among the three hydrogels with different ratios, the bonding strength of PEG-15% EPL-DA is the best. In addition, as the mass fraction of EPL-DA increases, the viscosity decreases. This is mainly due to the gradual increase in the number of amino groups in EPL-DA and the binding of more aldehyde groups, resulting in a decrease in the number of aldehyde groups binding to the tissue.

[0145] Test Example 4: Performance test of composite hydrogel

[0146] Sample: The composite hydrogel prepared in Example 2.

[0147] 1. Blood compatibility experiment

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

[0149] 1 mL of the solidified hydrogel sample was immersed in a mixed solution of 1 mL of RBC solution and 4 mL of PBS, and incubated in an incubator at 37 °C for 4 h and 8 h. Then, the solution was centrifuged at 3500 r / min for 5 min, and the absorbance of the supernatant at 540 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The hemolysis rate (H) was calculated according to Equation (2). At the same time, a positive control group and a negative control group were set up. The positive control group was a mixed solution of 1 mL of RBC and 4 mL of deionized water (hemolysis of blood in water), and the negative control group was a mixed solution of 1 mL of RBC solution and 4 mL of PBS.

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

[0151] In the above formula: Dt, Dnc, and Dpc are the absorbances of the test 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 in contact with materials. The better the blood compatibility of the material, the smaller the hemolysis rate. The hemolysis test results of the hydrogel samples prepared in this invention are as Figure 15 shown. The hemolysis rates of both hydrogel samples are lower than 5% of the industry standard, indicating that the hydrogel has good blood compatibility.

[0153] 2. In vitro degradation performance test

[0154] First, the hydrogel to be tested was freeze-dried and accurately weighed as W0. Then, the other test samples were immersed in PBS solution containing 0 or 1000 U / mL lysozyme and placed in a thermostatic shaker (37 °C, 70 rpm). At the measurement time point, the hydrogel was washed with ultrapure water, freeze-dried, and weighed as Wt. The weight retention rate of the hydrogel was calculated according to Equation (3) as follows:

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

[0156] The biodegradation performance 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 soaked in PBS buffer solution and 1000 U / mL lysozyme / PBS buffer solution for a certain period of time are as Figure 16 shown. After soaking in PBS solution for 6 days, the weight retention rate of the hydrogel was 39.3%, which was mainly due to the hydrolysis of ester bonds in the hydrogel, resulting in the degradation of the hydrogel. In the PBS buffer solution containing lysozyme, the hydrogel was completely degraded on the 8th day, indicating that the hydrogel had good biodegradability. The degradation rate of the hydrogel was relatively suitable for the tissue healing process. In the early stage of tissue healing, the hydrogel could maintain stability and antibacterial effect; then the hydrogel entered the disintegration stage and was gradually completely biodegradable, which would facilitate the subsequent removal of the hydrogel.

[0157] 3. In vitro drug release test

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

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

[0160] The liquid chromatography conditions were selected as acetic acid: water: methanol = 60:40:0.1, the flow rate was 1 mL / min, the injection volume was 20 μL, and the detection wavelength was 242 nm. A graph was plotted with the concentration as the abscissa and the peak area value as the ordinate, and linear regression was performed to obtain the standard curve equation. The prepared standard solutions were stored at 4°C.

[0161] The encapsulation efficiency and drug loading of liposomes in the composite hydrogel were determined by the ultrafiltration centrifugation method. Precisely measure 1 mL of the prepared sample solution and place it in an ultrafiltration centrifugal tube (cut-off relative molecular mass 4000). Centrifuge at 8000 r / min for 20 min at 4°C. Take 200 μL of the solution obtained after centrifugation in the outer tube of the ultrafiltration centrifugal tube, add 200 μL of methanol, and use a UV spectrophotometer to measure the concentration of α-ecdysone in the mixed solution. Calculate the content of ecdysone according to the standard curve to obtain the amount of free ecdysone. Calculate the encapsulation efficiency and drug loading of liposomes in the composite hydrogel according to the following formulas (4) and (5):

[0162] Encapsulation efficiency (%) = (the dosage of ecdysone during drug loading - the content of free ecdysone) / the dosage of melatonin during drug loading × 100% - Formula (4)

[0163] Drug loading rate (%) = the content of ecdysone in liposomes / the total mass of liposomes × 100% - Formula (5)

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

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

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

[0167] The microenvironment of human inflammatory tissues is a high ROS environment. By exploring the release degree of drugs in different environments, the characteristics of drug carriers and their effects on human tissues can be understood. The results are as Figure 18 shown. The release rate of α-ecdysone in the hydrogel is significantly greater under the condition of hydrogen peroxide than in the PBS environment. It is speculated that under the treatment of H 2 O 2 reactive oxygen species will trigger the rupture of liposomes.

[0168] To prove the rupture of liposomes triggered by reactive oxygen species under the treatment of H 2 O 2 , perform TEM observation on Lip@Ecdysone after incubating for 5 minutes in H 2 O 2 . The TEM image shows ( Figure 19 ), the spherical structure of Lip@Ecdysone incubated with H 2 O 2 is damaged, which indicates that H 2 O 2 causes the rupture of the diselenide bond in the liposome component, and then leads to the rupture of Lip@Ecdysone. Therefore, in the presence of hydrogen peroxide, the liposomes in the composite hydrogel will rupture, and then the release of α-ecdysone will be relatively faster; it is proved that the composite hydrogel prepared by the present invention has ROS responsiveness.

[0169] 4. Biocompatibility test

[0170] Add PMA to THP-1 cells and induce for 24 h. After adhesion, they become macrophages. Seed 5×10 3 cells / well in a 96-well plate, add the composite hydrogel sample to the cells, set a control group (without adding the composite hydrogel), and incubate at 37 °C, 5% CO 2Cultivate for 24, 72, and 120 h respectively in an incubator. Add 10 μL of CCK-8 reagent to each well at each time point, continue to incubate in the incubator for 4 h, and measure the absorbance at a wavelength of 450 nm using a microplate reader.

[0171] The results are as Figure 20 shown. The composite hydrogel was co-cultured with macrophages, had good biocompatibility, and the toxicity was at level 0-1.

[0172] 5. Small animal in vivo imaging experiment

[0173] Select rats with periodontitis models (periodontitis models were formed by removing the nylon wire ligating the second maxillary molar of rats after 1 week). Inject 40 μL of the sample labeled with FITC into the gingival tissue. Observe through the IVIS in vivo imaging system at 0, 1, 2, 3, 4, 5, 6, and 7 days.

[0174] The results are as Figure 21 shown. The fluorescence signal of the composite hydrogel sample was mainly concentrated at the injection site, and the signal could be maintained for 7 days; the signal gradually weakened but could always be detected, indicating that the composite hydrogel sample prepared by the present invention had good local retention and stability and was suitable for the treatment requirements of the periodontitis site.

[0175] 6. In vivo verification of targeting inflammatory macrophage mitochondria in gingival tissue

[0176] Select rats with periodontitis models. Inject 40 μL of the sample labeled with FITC into the gingival tissue. After 7 days, take the gingival tissue for sample fixation and ultra-thin sectioning, and use a transmission electron microscope (TEM) to observe the distribution of the composite hydrogel sample in macrophages.

[0177] The results are as Figure 22 shown. The sample (marked with a yellow arrow) was located inside the mitochondria (marked with a red arrow) of macrophages. At the same time, rough endoplasmic reticulum (marked with a blue arrow) and lysosomes (marked in white) were observed, indicating that the composite hydrogel sample successfully targeted the mitochondria of macrophages in gingival tissue. This result further verified the precise targeting delivery ability of the composite hydrogel and its effectiveness at the inflammatory site.

[0178] 7. In vivo safety evaluation

[0179] Inject 40 μL of the sample into the gingival tissue of rats with periodontitis models. Administer the drug every other day. After 4 consecutive weeks, take the main organs (heart, liver, spleen, lung, kidney) of the rats, fix, section, and perform HE staining to observe the toxicity and pathological changes of the composite material on the main organs. At the same time, set up a normal group and a periodontitis model group (lig is the model group) as controls.

[0180] The results are as Figure 23As shown. No obvious inflammation, necrosis or other pathological changes were observed in the tissue sections of the heart, liver, spleen, lung and kidney of the rats in each group. The main organ tissue structures were intact, similar to those of the normal group, and no toxic effects were observed. The results indicate that the composite hydrogel prepared by the present 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 rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced 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: The steps include: (1) 4-carboxybenzaldehyde, EDC and DMAP are mixed and dissolved in an organic solvent to obtain a mixed solution; under nitrogen, 4arm-PEG-OH is added to the mixed solution, stirred, washed, and dried to obtain 4arm-PEG-CHO, and PBS is added to obtain a 4arm-PEG-CHO solution; (2) adding water to polylysine and stirring evenly; then adding EDC and NHS to adjust the pH; adding dopamine hydrochloride in nitrogen, reacting in the dark, dialyzing, and freeze-drying to obtain EPL-DA; (3) dissolving DOTAP, lecithin, cholesterol, DSPE-Se-Se-PEG-NH2, DSPE-PEG-TPP, and DSPE-PEG-HA in an organic solvent, preparing cationic liposomes by a thin film dispersion method, and adding PBS to obtain a cationic liposome solution; (4) dissolving EPL-DA in a cationic liposome solution, vortexing, and ultrasonicating to obtain an EPL-DA solution; mixing the EPL-DA solution with a base, adding the 4arm-PEG-CHO solution, mixing, vortexing, and standing to obtain the composite hydrogel.

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

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

6. The composite hydrogel targeting macrophage mitochondria according to claim 5, characterized in that: The composite hydrogel targeting macrophage mitochondria further comprises an active drug loaded in the cationic liposome.

7. Use of the composite hydrogel targeting macrophage mitochondria as claimed in claim 5 or 6 in the preparation of a medicine or preparation for treating periodontitis.

8. The use according to claim 7, characterized in that The site of action of the drug or preparation is the tissue with subgingival lesions.

9. A medicine or preparation for treating periodontitis, characterized in that: The composite hydrogel targeting macrophage mitochondria according to claim 5 or 6.

10. The drug or preparation according to claim 9, characterized in that The dosage form of the preparation includes injection.

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