MaR1 nano-particles as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses MaR1 nano-particles as well as a preparation method and application thereof, and the MaR1 nano-particles are obtained by loading Maresin-1 into a polylactic acid nano-carrier. The MaR1 nano-particles are of a spherical structure, and the particle size ranges from 195 nm to 215 nm. The MaR1 nano-particles provided by the invention are beneficial to promoting the healing of diabetic wounds, inhibiting the formation of pathological scars and accelerating the regeneration and reconstruction of epidermis structures, so that the diabetic foot ulcer is treated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a MaR1 nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Diabetic foot ulcer (DFU) is one of the most serious complications of diabetes, and eventually may face the risk of amputation. Although the existing treatment methods (such as negative pressure suction, debridement, bacteriostasis, biological dressing, etc.) have made certain progress in the clinic, a considerable number of patients still face amputation due to further deterioration of the wound. Controlling wound infection and reducing inflammation have become the main treatment goal at present. Wound infection is caused by the disturbance of the transformation from the inflammatory phase to the proliferation phase, resulting in the disorder between pro-inflammatory and anti-inflammatory, eventually destroying the balanced environment required for normal wound healing, and forming the outcome of difficult wound healing. Therefore, it is of great significance to develop a product that is beneficial to promote diabetic wound healing. SUMMARY

[0003] In order to develop a product that promotes diabetic wound healing, the present application provides a MaR1 nanoparticle and a preparation method and application thereof. The MaR1 nanoparticle provided by the present application is beneficial to promote diabetic wound healing, inhibit pathological scar formation, and accelerate the regeneration and reconstruction of epidermal structure, thereby treating diabetic foot ulcer.

[0004] The present application provides a MaR1 nanoparticle, which is obtained by loading MaR1 into a polylactic acid nanocarrier.

[0005] The MaR1 nanoparticle provided by the present application is beneficial to promote diabetic wound healing, inhibit pathological scar formation, and accelerate the regeneration and reconstruction of epidermal structure, thereby treating diabetic foot ulcer.

[0006] Further, the MaR1 nanoparticle is in a spherical structure, and the particle size is 195nm-215nm.

[0007] The present application further provides a preparation method of the MaR1 nanoparticle, comprising the following steps: The left-handed polylactic acid solution is used as the oil phase, and the oil phase is mixed with the anhydrous ethanol solution containing Maresin-1 according to a volume ratio of 8-12:1 to obtain an oil phase mixture; then the water phase is added to the oil phase mixture, ultrasonic emulsification is performed to form a primary emulsion; the primary emulsion is mixed with a complex emulsion water phase to form a complex emulsion, and after the solvent is removed, the precipitate is collected by centrifugation to obtain the polylactic acid nanocarrier loaded with Maresin-1, i.e. the MaR1 nanoparticle.

[0008] Further, the volume ratio of the oil phase and the anhydrous ethanol solution containing Maresin-1 (drug-containing organic phase) is 10:1.

[0009] Further, the volume ratio of the oil phase to the water phase is 1:5-15.

[0010] Further, the volume ratio of the oil phase to the water phase is 1:10.

[0011] Further, the water phase is 1% (w / v) polymethyl methacrylate.

[0012] Further, the volume ratio of the initial emulsion to the re-emulsion water phase is 1:20-40.

[0013] Further, the concentration of the levorotatory polylactic acid solution is 50 μg / ml-500 μg / ml.

[0014] Further, the solvent of the levorotatory polylactic acid solution is dichloromethane.

[0015] Further, the re-emulsion water phase is 0.5% (w / v) polymethyl methacrylate.

[0016] The application further provides application of the MaR1 nanoparticle in preparation of a medicine for treating diabetic foot ulcers.

[0017] Further, the MaR1 nanoparticle is used for preparation of a medicine for promoting healing of diabetic wounds.

[0018] Further, the medicine has the following functions: anti-inflammation; promoting wound healing; inhibiting pathological scar formation; accelerating regeneration and reconstruction of epidermal structure.

[0019] Further, the medicine is a MaR1 NPs solution obtained by dissolving MaR1 NPs in PBS.

[0020] Compared with the prior art, the application has the following beneficial effects: The application prepares a MaR1 nanoparticle with uniform particle size, good stability and no significant biological toxicity, which promotes repair of an epidermal barrier and collagen ordered remodeling, inhibits pathological scar formation, and thus restores a skin tissue structure homeostasis. The MaR1 nanoparticle has the following functions: anti-inflammation; promoting wound healing; inhibiting pathological scar formation; accelerating regeneration and reconstruction of epidermal structure.

[0021] The MaR1 nanoparticle promotes healing of a wound of a diabetic mouse through an NF-κB / STAT3 signal pathway, and down-regulates expression of inflammatory factors. The MaR1 nanoparticle significantly inhibits M1 polarization of inflammatory macrophages, up-regulates MERTK expression through regulation of the NF-κB / STAT3 signal pathway, and thus enhances the cell corpse effect of the macrophages. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only need to be some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative work on the premise of not paying any creative work.

[0023] Figure 1 Influence of different preparation processes on particle size and encapsulation efficiency of nanoparticles; experimental data are expressed as mean ± SD (n≥3), and the statistical significance determination criteria are: **** p <0.0001; In the figure, A is the particle size (nm) of the nanoparticles under the volume ratio of the oil phase to the water phase; B is the particle size (nm) of the primary emulsion: double emulsion water phase nanoparticles under different volume ratios; C is the standard curve of MaR1 measured by HPLC; D is the encapsulation efficiency (%) of the PLA and MaR1 solution under different proportions.

[0024] Figure 2 Characterization analysis of MaR1 NPs prepared in Example 1 of the present application; data are expressed as mean ± SD (n≥3); In the figure, A is the intensity distribution diagram of the particle size analysis measured by Malvern laser diffraction; B is the comparison of the Zeta potential (mV) of empty NPs and MaR1 NPs; C is the number distribution diagram of the particle size analysis measured by Malvern laser diffraction; D is the detection result of the particle size stability (nm) of empty NPs and MaR1 NPs.

[0025] Figure 3 Image scanning analysis of MaR1 NPs; experimental data are expressed as mean ± SD (n≥3); In the figure, A is the transmission electron microscopy (TEM) diagram of empty NPs and MaR1 NPs, and the scale = 5.0 µm; B is the scanning electron microscopy (SEM) diagram of empty NPs and MaR1 NPs. Scale = 2.0 µm / 200 nm.

[0026] Figure 4 Safety verification of MaR1 NPs in vivo and in vitro; experimental data are expressed as mean ± SD (n≥3); In the figure, A is the cell activity detected by CCK-8 experiment under different concentrations of empty NPs; B is the cell activity of MaR1 NPs with different concentrations detected by CCK-8 experiment C is the H&E staining result of internal organs after MaR1 NPs intervention, scale = 400 μm.

[0027] Figure 5 The in vivo experimental design and diabetic wound model detection; experimental data are expressed as mean ± SD (n≥3; In the figure, A is the flowchart of the mouse in vivo experiment; B is the fasting blood glucose (FBG) level of mice (mmol / L); C is the weight change of mice (g).

[0028] Figure 6 The effect of different treatment groups on the wound healing of diabetic mice; experimental data are expressed as mean ± SD (n≥3), and the statistical significance determination criteria are: p <0.01; In the figure, A is a representative picture of the wound healing process of diabetic mice; scale = 1 cm; B is the change of wound area of mice within 14 days (cm 2 ); C is the statistical result of wound healing of diabetic mice after drug intervention in each group (%).

[0029] Figure 7 The effect of different treatment groups on tissue remodeling of diabetic mice; experimental data are expressed as mean ± SD (n≥3), and the statistical significance determination criteria are: p > 0.05, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 (vs. DWM group); In the figure, A is the H&E staining and Masson staining result of diabetic wound of mice; scale = 50 μm; Es: eschar; NE: new epidermis; GT: regenerating granulation tissue; B is the statistical result of granulation tissue thickness; C is the statistical result of epidermis thickness; D is the statistical result of collagen deposition.

[0030] Figure 8 The effect of different treatment groups on the expression of inflammatory factors in the wound of diabetic mice; experimental data are expressed as mean ± SD (n≥3), and the statistical significance determination criteria are: p > 0.05, *p <0.05, ** p <0.01,*** p <0.001, **** p <0.0001; In the figure, A represents the mRNA expression of IL-1β, IL-6, TNF-α, iNOS, and MERTK in the skin of mice in different treatment groups (data were log-transformed (Log2) for statistical analysis). B shows the mRNA expression of TNF-α, iNOS, and MERTK in the skin of mice in different treatment groups; C represents the ELISA results (ng / mL) of IL-1β, IL-6 and TNF-α in serum of different treatment groups (data were logarithmically transformed (Log10) for statistical analysis).

[0031] Figure 9 Immunohistochemical analysis of wound inflammatory factor expression in different treatment groups; experimental data are expressed as mean ± SD (n≥3), statistical significance criteria: ns p >0.05, * p <0.05, ** p <0.01, *** p <0.001,**** p <0.0001 (vs. DWM group); In the figure, A shows the immunohistochemical staining results of IL-1β, IL-6 and TNF-α in the wound tissue of different treatment groups 14 days after surgery. Scale bar = 100 μm. B represents the average optical density of the measured field of view. The staining intensity was quantified and statistically analyzed (immunohistochemical staining area). From left to right, the staining intensity represents IL-1β, IL-6, and TNF-α.

[0032] Figure 10 The effects of different treatment groups on epidermal regeneration of wounds in diabetic mice were investigated. Data are expressed as mean ± standard deviation (SD) (n ≥ 3) and statistical analysis was performed. p >0.05, ** p <0.01, *** p <0.001, *** p <0.0001 (vs. DWM group); In the figure, A shows the CK14 / CK10 immunofluorescence staining results of wound tissues from diabetic mice in different treatment groups, with scale bar = 20 μm; B represents the quantitative analysis of CK14 positive expression in wound tissues of diabetic mice in different treatment groups; C is the quantitative analysis of CK10 positive expression of wound tissue of different treatment groups of diabetic mice. DETAILED DESCRIPTION

[0033] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of the present application is not limited to the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts are within the scope of the present application. The experimental methods described in the embodiments of the present application are conventional methods, and the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0034] Example 1: MaR1 nanoparticles and preparation method and application thereof.

[0035] MaR1 is Maresin-1 of Cayman brand (USA).

[0036] I. MaR1 nanoparticles and preparation method thereof 1. Preparation steps of MaR1 nanoparticles In this experiment, nanoparticles were prepared by modified oil-in-water (o / w) emulsion solvent evaporation technology.

[0037] Dissolve 150 μg of L-polylactic acid (PLA) in 2 ml of dichloromethane to form a clear and transparent solution, and obtain a PLA solution as an oil phase. Take 100 μL of the PLA solution and place it in a pre-cooled beaker, then quickly add 10 μL of MaR1 anhydrous ethanol solution (referred to as MaR1 solution) and mix thoroughly, then add 1 mL of 1% (w / v) PEMA (poly (methyl methacrylate)) as an aqueous phase, and emulsify under 300 W ultrasonic conditions at 0°C ice bath for 40 s to obtain a primary emulsion (oil-in-water emulsion), forming a primary emulsion phase. Slowly add 1 mL of the primary emulsion to 40 mL of 0.5% (w / v) PEMA (poly (methyl methacrylate)) aqueous solution to form a re-emulsion, and the 0.5% (w / v) PEMA aqueous solution is used as the re-emulsion aqueous phase. Stir with a magnetic stirrer in a fume hood for 6 h, and after the dichloromethane is completely volatilized, place the mixed suspension of the formed nanoparticles in a high-speed centrifuge (4°C, 12000 rpm, 30 min) to collect the MaR1 nanoparticle precipitate. Discard the supernatant, resuspend the MaR1 nanoparticle precipitate with enzyme-free water, and centrifuge again (4°C, 12000 rpm, 15 min). This washing process is repeated three times. After dehydration and drying in a freeze dryer, MaR1 nanoparticles (MaR1 NPs) are obtained, and stored at -20°C.

[0038] 2. Effect of different preparation systems on particle size distribution and drug encapsulation efficiency of prepared nanoparticles (1) Effect of different volume ratios of oil phase: water phase on the particle size (nm) of obtained nanoparticles The volume ratio of oil phase: water phase was set as: 1:1, 1:5, 1:10, 1:15 and 1:20.

[0039] According to the above oil phase: water phase ratio and the preparation steps of MaR1 nanoparticles described above, MaR1 nanoparticles were prepared under different oil phase: water phase ratios, and the effect of different volume ratios of oil phase: water phase on the particle size (nm) of the prepared nanoparticles was analyzed.

[0040] (2) Effect of different volume ratios of primary emulsion: re-emulsion water phase on the particle size (nm) of nanoparticles The volume ratio of primary emulsion: re-emulsion water phase was set as: 1:5, 1:10, 1:20, 1:40 and 1:80.

[0041] According to the above volume ratio of primary emulsion: re-emulsion water phase and the preparation steps of MaR1 nanoparticles described above, MaR1 nanoparticles were prepared under different volume ratios of primary emulsion: re-emulsion water phase, and the effect of different volume ratios of primary emulsion: re-emulsion water phase on the particle size (nm) of the prepared nanoparticles was analyzed.

[0042] (3) Effect of different volume ratios of PLA solution and MaR1 solution on the encapsulation efficiency (%) of obtained nanoparticles The volume ratio of PLA solution and MaR1 solution was set as: 1:1, 5:1, 10:1, 15:1 and 20:1.

[0043] According to the above different volume ratios of PLA solution and MaR1 solution and the preparation steps of MaR1 nanoparticles described above, MaR1 nanoparticles were prepared under different volume ratios of PLA solution and MaR1 solution, and the effect of different volume ratios of PLA solution and MaR1 solution on the encapsulation efficiency (%) of obtained MaR1 nanoparticles was analyzed.

[0044] The calculation process of encapsulation efficiency is as follows: a. Prepare MaR1 standard curve: Take 1 μL of MaR1 anhydrous ethanol solution, dilute its concentration to 0.002 ng / mL, 0.005 ng / mL, 0.01 ng / mL, 0.02 ng / mL, 0.04 ng / mL with anhydrous ethanol, and measure the peak area at a wavelength of 272 nm in a high performance liquid chromatograph. The measured data is plotted as a standard curve of peak area-concentration.

[0045] b.Determination of MaR1 NPs encapsulation efficiency: The content of MaR1 loaded into the nanoparticles was calculated by a direct method. The prepared MaR1 NPs were placed in 500 μL dichloromethane, and after ultrasonic demulsification (60 W, a total of 20 S), they were placed on a magnetic stirrer in the dark. After the dichloromethane was completely volatilized, 200 μL anhydrous ethanol was added to extract MaR1, and the mixture was magnetically stirred at room temperature for 20 min. The supernatant was collected by high-speed centrifugation (4 ℃, 12000 rpm, 30 min). The peak area was measured at 272 nm by high performance liquid chromatography (HPLC), and the MaR1 content was calculated according to the standard curve. The encapsulation efficiency (EE) and drug loading rate (DL) of MaR1 NPs were calculated by the following formula:

[0046] Encapsulation efficiency EE (%) = (mass of encapsulated MaR1 / mass of total added MaR1) x 100%.

[0047] To accurately quantify the drug loading efficiency of the nanoparticles, the present application established a high performance liquid chromatography (HPLC) analysis method. The calibration curve of MaR1 concentration and integral area established by a series of MaR1 standard samples showed a good linear relationship (R² = 0.999) (Fig. 1C). Figure 1 Based on this, the effects of different PLA / MaR1 contents on the encapsulation efficiency were systematically evaluated.

[0048] The results are shown in Figure 1 When the volume ratio of oil phase to water phase (O:W) was 1:10, the prepared nanoparticles had the optimal particle size distribution (212.9 ± 3.48 nm, Fig. 2A), and the polydispersity index (PDI) was less than 0.2, indicating good uniformity of particle size. Figure 1 When the volume ratio of primary emulsion to water phase of re-emulsion was 1:40, the average particle size of the prepared nanoparticles was significantly reduced to 204.18 ± 3.57 nm (Fig. 2B), and the zeta potential measurement showed that the surface charge stability was enhanced. Figure 1 When the volume ratio of PLA solution to MaR1 solution was 10:1, the highest encapsulation efficiency (37.57 ± 2.02%) was obtained (Fig. 2D), which was significantly higher than that of other ratio groups (Fig. 2D). Figure 1 p <0.0001>.

[0049] In summary, the present application successfully prepared MaR1 nanoparticles, and found that when the volume ratio of oil phase to water phase was 1:10, the volume ratio of primary emulsion to water phase of re-emulsion was 1:40, and the volume ratio of PLA to MaR1 was 10:1, the prepared nanoparticles not only had ideal particle size distribution and surface characteristics, but also ensured high drug encapsulation efficiency, providing a reliable pharmaceutical basis for subsequent in vivo pharmacodynamic evaluation and mechanism of action research.

[0050] II. Characterization and analysis of MaR1 nanoparticles​ In this invention, MaR1 nanoparticles (MaR1 NPs) were prepared with an oil-to-water phase volume ratio of 1:10, a primary emulsion-to-secondary emulsion aqueous phase ratio of 1:40, and a PLA-to-MaR1 volume ratio of 10:1. As a basis for subsequent research, the prepared MaR1 nanoparticles were systematically characterized to evaluate their physicochemical properties and stability.

[0051] Preparation of empty nanoparticles (empty NPs): Weigh 150 μg of polylactic acid (PLA) and dissolve it in 2 ml of dichloromethane. After complete dissolution, a clear and transparent solution is formed, which is the PLA solution and is used as the oil phase. Take 100 μL of PLA solution and place it in a pre-cooled beaker. Then, quickly add 10 μL of anhydrous ethanol solution and mix thoroughly. Then, add 1% (w / v) PEMA, which is used as the aqueous phase. Ultrasonic emulsify at 300 W for 40 s under ice bath conditions to obtain the primary emulsion (oil-in-water emulsion), forming the primary emulsion phase. 1 mL of colostrum was slowly added dropwise to 40 mL of 0.5% (w / v) PEMA aqueous solution to form a secondary emulsion. The 0.5% (w / v) PEMA aqueous solution served as the aqueous phase of the secondary emulsion. The mixture was stirred with a magnetic stirrer in a fume hood for 6 h. After the dichloromethane had completely evaporated, the resulting nanoparticle suspension was centrifuged in a high-speed centrifuge (4 °C, 12000 rpm, 30 min) to collect the nanoparticle precipitate. The supernatant was discarded, and the nanoparticle precipitate was resuspended in enzyme-free water and centrifuged again (4 °C, 12000 rpm, 15 min). This washing process was repeated three times. After dehydration and drying using a freeze dryer, empty nanoparticles (empty NPs) were obtained and stored at -20 °C.

[0052] This invention employs dynamic light scattering (DLS) technology to determine the particle size distribution, scattering intensity, and particle number distribution of empty nanoparticles (empty NPs) and MaR1 nanoparticles (MaR1 NPs). The specific steps are as follows:

[0053] Particle size measurement: The prepared particles were uniformly dispersed in enzyme-free water and ultrasonically dispersed in an ultrasonic cleaner for 30 seconds. After thorough mixing, 1.2 mL of the suspension was taken and its particle size distribution was measured in a Malvern particle size analyzer. Water was selected as the dispersed phase, the temperature was 25 ℃, and full wavelength scanning was performed. The average particle size of the PdI<0.03 array was calculated.

[0054] The results are as follows Figure 2 As shown, the scattering intensity distribution of the nanoparticles exhibits a single-peak pattern. Figure 2 The peak width was relatively narrow, mainly concentrated in the range of 200 nm to 220 nm. Further analysis of the particle number distribution revealed that approximately 40% of the particles were concentrated around 200 nm.Figure 2 C). All of the above verify that the nanoparticles have good particle size uniformity (PDI < 0.3).

[0055] Zeta potential is a key indicator for evaluating the colloidal stability of nanoparticles. The measurement of zeta potential is as follows: the prepared nanoparticles are ultrasonically dispersed for 30 s at the original concentration. The potential sample cell is first immersed in anhydrous ethanol for 5 s, then washed twice with enzyme-free water for standby. The zeta potential sample cell is filled with the sample using a 1 mL syringe, completely covering the electrodes and without air bubbles, and then placed in a Malvern particle size analyzer, with the measurement selecting zeta potential: solvent water, temperature 25℃, repeated detection 100 times to calculate the average value.

[0056] The experimental results show that the zeta potential of empty NPs is -46.76 ± 1.36 mV, and the absolute value of the zeta potential of MaR1 NPs is slightly lower than that of empty NPs (-44.85 ± 1.69 mV). Figure 2 B). Under the storage condition of 4℃, the present application carries out a 14-day stability test, and the results show that the particle size of the two kinds of NPs does not change significantly, and the PDI value is always maintained below 0.3 ( Figure 2 D).

[0057] In summary, the above characterization results, the successfully prepared empty NPs and MaR1 NPs both have reasonable particle size distribution, high surface charge stability and good long-term storage performance, which provides a reliable nano-preparation basis for subsequent in vitro and in vivo experiments.

[0058] III. Image scanning analysis of MaR1 NPs After successfully constructing MaR1 nanoparticles with good stability, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are further used to characterize the morphology of the nanoparticles.

[0059] Transmission electron microscopy: after mixing and ultrasonicating the MaR1 NPs for 5 min, the nanoparticles are loaded on a 300 mesh carbon film copper mesh. After being washed with distilled water, 2% phosphotungstic acid sodium staining solution is added for staining (2 times, 10 s each time). After the stained copper mesh is completely dried, imaging observation is carried out on a transmission electron microscope at an acceleration voltage of 80 kV.

[0060] Scanning electron microscopy: 50 μL of MaR1 NPs suspension is dropped on a glass slide treated with anti-dropping agent and dried at room temperature; the sample is tightly attached to the conductive carbon film double-sided tape, and then sprayed with gold in an ion sputtering instrument for about 30 s. The sample is observed under a scanning electron microscope and the picture is collected.

[0061] TEM results show that: empty NPs and MaR1 NPs present regular spherical morphology, clear edge (Fig. 1A). Figure 3 The SEM image further verifies that empty NPs and MaR1 NPs both present uniform spherical structure, smooth surface, and the particle size distribution range is 195nm-215nm (Fig. 1B). Figure 3 And the electron microscope observation result is highly consistent with the particle size measured by dynamic light scattering, and this uniform morphology is conducive to enhancing the stability of the drug carrier.

[0062] Four, in vitro and in vivo safety verification of MaR1 NPs MaR1 NPs prepared according to the conditions in the first step of Example 1: the volume ratio of oil phase and water phase is 1:10, the ratio of primary emulsion and re-emulsion water phase is 1:40, and the volume ratio of PLA and MaR1 is 10:1, and the empty NPs prepared in Example 1 are used as experimental objects for subsequent experimental research.

[0063] MaR1 NPs are prepared into MaR1 NPs solutions with concentrations of 2.3 mg / mL, 4.61 mg / mL, 9.22 mg / mL and 18.4 mg / mL using DMEM medium containing 10% FPS. The empty NPs are prepared into empty NPs solutions with concentrations of 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL and 0.5 mg / mL using the medium.

[0064] The present application analyzes the biological safety of MaR1 NPs solution and empty NPs solution with different concentrations from in vitro cytotoxicity experiment and in vivo pathological evaluation. In vitro experiment uses CCK-8 method to detect the influence of different concentrations of empty NPs solution and MaR1 NPs solution on RAW264.7 cell activity. In vivo safety evaluation mainly carries out H&E staining on important organs (including heart, liver, lung, spleen and kidney tissues).

[0065] 1. In vitro experiment CCK-8 method for detecting nanoparticle biological toxicity RAW264.7 cells are inoculated in a 96-well plate (about 1×10 4 cells per well), 100 μL of complete culture medium is added to each well (6 duplicate wells are set for each group), and the cells are cultured for 18 h until they are completely attached and evenly distributed. A circle of 100 μL PBS buffer is added around the cells to prevent evaporation. The experiment is divided into 5 groups, and the experimental grouping is as follows:

[0066] Blank group: no cells, only medium; denoted as NC; Positive control group: cells + medium; denoted as DWM; Empty NPs experimental group: cells + Empty NPs solution; MaR1 NPs experimental group: cells + MaR1 NPs solution.

[0067] Different concentrations of nanoparticle solutions were added to the experimental groups, and the culture was continued for 24 h. 10 μL of CCK-8 reagent was added to each well (without changing the culture medium), and the mixture was gently mixed and incubated for 1 h. The OD value of each well was measured at 450 nm using a microplate reader (OD 450 ), and the experimental results were used to calculate the cell survival rate.

[0068] Cell survival rate (%) = (experimental group OD 450 value - blank group OD 450 value) / (control group OD 450 value - blank group OD 450 value) x 100%.

[0069] The results are shown in Figure 4 : even after 24 hours of high concentration intervention, the cell survival rate of the two kinds of nanoparticles was still more than 80% (A of Figure 4 and B of Figure 4 ).

[0070] The in vivo safety evaluation results showed that the organ tissue structure of each group was complete, and no pathological changes such as necrosis and fibrosis were found (C of Figure 4 ). In summary, MaR1 NPs showed good biocompatibility in vitro and in vivo, and is a kind of nanomaterial with good biological safety.

[0071] Five, the therapeutic effect of MaR1 NPs on wound healing in diabetic mice 1. Construction of a wound model in diabetic mice (1) Construction of a diabetic model Six-week-old male C57BL / 6J mice were purchased and allowed to freely eat and drink water. After 1 week of adaptive feeding, the experiment began. Subsequently, the mice were fed a 60% high-fat (cholesterol) diet for 2 months until their body weight reached 30 g. After fasting for 12 h, the mice were intraperitoneally injected with a streptozotocin (STZ) solution (0.1 M citric acid buffer, prepared fresh, placed on ice, and strictly protected from light) at a dose of 50 mg / kg. After injection, the mice were given a normal diet, and the injection was continued for 7 days. After 7 days, blood was collected from the tail vein, and the fasting blood glucose was measured. If the blood glucose value was ≥16.8 mmol / L for 3 consecutive times and was accompanied by symptoms such as polydipsia, polyphagia, polyuria, and weight loss ("three more and one less"), the diabetic mouse model was considered to have been successfully established, and the diabetic mouse model was denoted as DM.

[0072] (2) Diabetic wound model construction: One week after the successful modeling of diabetic mice, the back was depilated one day in advance. The mice were anesthetized (1% sodium pentobarbital intraperitoneal injection, 60 mg / kg), and the skin was routinely disinfected and laid. A 10 mm diameter puncher was used to construct a full-thickness skin defect wound on the back of the mouse to obtain the wound model mouse (DWM). After punching, the wound was protected with a clean dressing to avoid infection and adhesion of the wound caused by mouse activity. Subsequently, the wound model mice were divided into a model group (DWM), an empty NPs experimental group, a MaR1 experimental group and a MaR1 NPs experimental group, and a healthy mouse blank control group (NC). The blank control group (NC) and the model group were intraperitoneally injected with 100 microliters of normal saline, and the experimental groups were intraperitoneally injected with 200 ng of each group of drugs per mouse and treated for 14 days. PBS dissolved MaR1 NPs to obtain MaR1 NPs solution, PBS dissolved empty NPs to obtain empty NPs solution. PBS dissolved Maresin 1 to obtain MaR1 solution.

[0073] (3) Wound healing evaluation: On the 0th, 2nd, 7th, 10th and 14th day after the operation, the wound healing condition was collected, and the wound size was measured by ImageJ software to calculate the wound healing rate.

[0074] Wound healing rate (%) = (initial wound area - current wound area) / initial wound area x 100%.

[0075] The results show that the fasting blood glucose (FBG) level of the diabetic group mice is significantly increased, reaching more than 16.8 mmol / L ( Figure 5 B), and the body weight of the mice decreases significantly within 4 weeks ( Figure 5 C), which indicates that the diabetic model has been successfully constructed.

[0076] 2, MaR1 NPs accelerates wound healing of diabetic mice The present application records the healing condition of the wounds of the mice in each group within 14 days after the operation, and performs statistical analysis.

[0077] The results show that: the skin wounds of the NC group and the MaR1 NPs group are smooth and flat, and no scab is observed; the wounds of the DWM group are obviously red, the scab is not detached, and are in an ulcer state; a small amount of red scab is observed on the wounds of the empty NPs group; white scab marks are observed on the wounds of the MaR1 group ( Figure 6 A). The wound area of the mice gradually decreases with the extension of time ( Figure 6 B), and the wound healing rate of the mice treated with empty NPs, MaR1 and MaR1 NPs is significantly better than that of the DWM group mice ( Figure 6 C). The above results show that MaR1 NPs can significantly promote the wound healing of diabetic mice.

[0078] 3. MaR1 NPs improve skin tissue remodeling in diabetic mice In order to observe the wound healing of mice in each group from a microscopic point of view, the skin of the wound of the mice was taken on the 14th day after the operation for longitudinal section and pathological staining.

[0079] Paraffin section preparation: The fixed tissue was placed in an automatic dehydrator (including dehydration and transparency process), and the tissue after immersion in wax was placed in a mold, embedded, and made into a wax block; the section with a thickness of 5 μm was cut, spread in warm water at 40 ℃, attached to a glass slide, and baked in a 37 ℃ oven for 1 h. The wax block and section were stored in a 4 ℃ refrigerator.

[0080] Pathological staining: hematoxylin-eosin (HE) staining: the tissue section was sequentially subjected to deparaffinization (xylene 2 times, 10 min) and hydration (100%, 95%, 80%, 70% ethanol, 5 min); the section was stained in hematoxylin dye for 5 min, washed with running water for 5 min; the section was immersed in eosin dye for 1 min, washed with running water for 30 s; the section was sequentially dehydrated in ethanol (70%, 80%, 90%, 95%, 100%, 5 min); then transparentized with xylene (I, II, 2 times, 5 min); finally, the section was mounted with neutral resin, dried at room temperature for 48 h, and then the image was collected by a digital scanner.

[0081] Masson staining: after the mouse skin tissue section was deparaffinized and hydrated, the staining was completed according to the procedure of the Masson staining kit, then the section was mounted with neutral resin, dried at room temperature for 48 h, and then the image was collected by a digital scanner.

[0082] Immunohistochemistry: (1) deparaffinization and hydration: deparaffinization and hydration to distilled water according to the procedure of HE staining. (2) antigen repair and blocking: the section was immersed in sodium citrate buffer at pH 6.0, heated at 95 ℃ for 15 min, and cooled to room temperature. After the liquid was absorbed, 5% BSA blocking solution was added, and incubated at room temperature for 30 min. (3) primary antibody incubation: the diluted primary antibody was added, and incubated at 4 ℃ overnight, and washed with PBS (3 times, 5 min). (4) secondary antibody incubation: the secondary antibody was added, and incubated at room temperature on a shaking table for 1 h, and washed with PBS (3 times, 5 min). (5) color development: DAB color developing solution was added, and the color development time was controlled under a microscope, and the reaction was terminated with distilled water. Hematoxylin was added for 1 min, and washed with running water for 10 min. (6) dehydration, transparency and mounting: mounted with neutral resin, dried at room temperature for 48 h, and then the image was collected.

[0083] Paraffin section immunofluorescence staining: (1) De-waxing and hydration: de-waxing and hydration to distilled water according to the HE staining procedure. (2) Antigen repair and blocking: immerse the section in pH 6.0 sodium citrate buffer, heat at 95 ℃ for 20 min, and cool to room temperature. Add 5% BSA blocking solution dropwise, and incubate at room temperature for 30 min. (3) First antibody (Ab1) incubation: 1% BSA diluted first antibody is incubated at 4 °C overnight, PBS washing (3 times, 5 min); fluorescently labeled secondary antibody matched with Ab1 species (37 ℃ incubation for 1 h), PBS washing (3 times, 5 min); 4% paraformaldehyde room temperature fixation for 10 min, PBS washing (2 times, 5 min). Use unlabelled anti-mouse Fab fragments, incubate at room temperature for 30 min to block the remaining binding sites of the first round of antibodies, and PBS washing (2 times, 5 min). (4) Second antibody (Ab2) incubation: 1% BSA diluted second antibody is incubated at 4 °C overnight, PBS washing (3 times, 5 min); fluorescently labeled secondary antibody with a different wavelength from Ab1 (37 ℃ incubation for 1 h), PBS washing (3 times, 5 min). (5) Nucleus staining: DAPI is incubated at room temperature for 5 min, and PBS washing (3 times, 5 min). (6) Mounting and observation: add anti-fluorescence quenching mounting agent dropwise, dry with a cover glass, and store in the dark. According to the fluorescent label, select the appropriate excitation wavelength to collect images.

[0084] The present application uses Graphpad Prism 9.5.1 software for statistical analysis and making statistical charts, and the data results are expressed as mean ± standard deviation (Mean ± SD). The differences between two groups are compared by t test, and the comparison among multiple groups is compared by One-Way ANOVA and variance homogeneity test. When P <0.05 indicates that the difference is statistically significant.

[0085] The HE staining results show that: compared with the NC group, the epidermis of the DWM group is significantly thinned, the granulation tissue is significantly reduced, the basal cell layer is arranged in disorder, and is accompanied by local keratin layer shedding; while in the NC group and the MaR1 NPs group, the diameter of the wound of the diabetic mice is significantly reduced, the epidermis is significantly increased, the basal cell layer is arranged in order, and part of the hair follicle structure is regenerated in the dermis, indicating that the repair of the epidermal barrier. Figure 7 A of FIG. 1, Figure 7 B of FIG. 1, and Figure 7 C of FIG. 1).

[0086] Masson staining results showed that: the dermal collagen fibers in the DWM group were arranged in disorder and distributed in fragments, and the proportion of collagen fiber area was significantly lower than that in the NC group, while the collagen deposition in the MaR1 NPs group was significantly improved; in addition, the newly formed collagen in the DWM group was mainly composed of immature collagen with light blue color, accompanied by local dense deep blue fiber bundles, indicating pathological scar formation; while the collagen fibers in the MaR1 NPs group were arranged in parallel, and the mature collagen proportion was significantly increased Figure 7 A of the application and Figure 7 D of the application.

[0087] In summary: MaR1 NPs can inhibit pathological scar formation by promoting epidermal barrier repair and collagen remodeling, thereby restoring the skin tissue structure homeostasis.

[0088] 4, MaR1 NPs inhibit the expression of inflammatory factors in the wound of diabetic mice In order to clarify the remodeling effect of MaR1 NPs on the inflammatory microenvironment of diabetic wound, the expression levels of related inflammatory factors and cell markers were systematically analyzed by qPCR and ELISA technology.

[0089] Firstly, the RNA of the wound tissue of the MaR1 treatment group in the pre-experiment was extracted, and the expression of related inflammatory factors and cell markers was detected by qPCR. The related inflammatory factors and the primer information used in qPCR are shown in Table 1.

[0090] Table 1 Related inflammatory factors and primer information used in qPCR The results showed that: MaR1 treatment significantly down-regulated the expression levels of inflammatory factors such as TNF-α, IL-1β and IL-6 in the wound tissue of the DWM group; and inhibited the expression of M1 type macrophage marker (iNOS), while up-regulated the expression of macrophage efferocytosis related gene (MERTK) (A of the application). This indicates that MaR1 can not only inhibit the pro-inflammatory response, but also promote wound healing by enhancing the efferocytosis function of macrophages. Figure 8

[0091] Therefore, the RNA of the wound tissue of the mouse treated with MaR1 NPs for 14 days was further extracted, and the expression levels of TNF-α, iNOS and MERTK were detected (B of Figure 8). The results showed that the MaR1 NPs group and the simple MaR1 group showed consistent trends in terms of inhibition of inflammatory factors and up-regulation of efferocytosis related genes, although there was no significant difference between the two groups in statistics. This result suggests that MaR1 NPs and free MaR1 have similar molecular regulation effects to a certain extent. ​

[0092] To further verify the systemic anti-inflammatory effect of MaR1 NPs, the expression levels of inflammatory factors in mouse serum were detected. The results showed that, compared with the DWM group, MaR1 NPs significantly reduced the levels of TNF-α, IL-1β, and IL-6 in mouse serum, and the differences were statistically significant (Figure 8, C, ****). p The value <0.0001 indicates that it has a good systemic anti-inflammatory effect in vivo, and the effect is better than that of the drug alone.

[0093] In summary, MaR1 NPs treatment helps modulate the inflammatory microenvironment of wounds in diabetic mice and may promote wound repair by promoting macrophage phenotypic transformation and enhancing phagocytic function.

[0094] 5. Immunohistochemical analysis of the regulation of wound inflammatory factor expression by MaR1 NPs In this invention, skin tissue from wounds of mice in different treatment groups was extracted and further stained with IHC.

[0095] The results showed that on postoperative day 14, the expression levels of IL-1β, IL-6, and TNF-α in the MaR1 and MaR1 NPs groups were significantly lower than those in the DWM group. Figure 9 A and Figure 9 (B). Positive areas were mainly concentrated at the wound edge, indicating that MaR1 NPs intervention had a better effect on local inflammation suppression at the wound edge; in terms of tissue repair, the epidermis and dermis of mice in the DWM group were not completely fused, and there was obvious separation between the two, indicating incomplete wound repair; while the wounds of the MaR1 NPs group, empty NPs group and free MaR1 group showed complete fusion of epidermis and dermis, and there were a large number of newly generated hair follicles in the dermis. Figure 9 The results (A) indicate significant tissue repair and remodeling. These results further demonstrate that MaR1 NPs have a significant advantage in promoting wound inflammation control and tissue repair.

[0096] 6. MaR1 NPs promote epidermal differentiation in diabetic mice This invention uses multiplex fluorescence technology to evaluate the regulatory role of MaR1 NPs in epidermal regeneration of diabetic wounds by epidermal differentiation markers (CK14 / CK10).

[0097] The results showed that the CK14⁺ basal cell density was decreased in the DWM group, accompanied by a reduction in the thickness of the CK10⁺ differentiation layer (vs. NC group), suggesting an imbalance in epidermal homeostasis and impaired barrier function (Figure 10, A and B). Figure 10B); the CK14+ cell density of the MaR1 NPs group recovered to near physiological level, and the CK10+ layer thickness was also significantly improved (vs the DWM group) Figure 10 A and Figure 10 C). The up-regulation of the synergistic expression of CK14 and CK10 indicates that the MaR1 NPs can accelerate the regeneration and reconstruction of the epidermal structure by bidirectional regulation of the basal cell proliferation activity and the terminal differentiation process.

[0098] It should be noted that the empty NPs and the Empty NPs in the present application represent the same object.

[0099] Although preferred embodiments of the present application have been described, those skilled in the art who understand the basic inventive concept can make additional changes and modifications to these embodiments.

[0100] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A MaR1 nanoparticle, characterized in that, The MaR1 nanoparticles were obtained by loading the drug Maresin-1 onto a polylactic acid nanocarrier.

2. The MaR1 nanoparticles according to claim 1, characterized in that, The MaR1 nanoparticles have a spherical structure and a particle size of 195 nm to 215 nm.

3. A method for preparing MaR1 nanoparticles according to claim 1 or 2, characterized in that, Includes the following steps: Using a L-type polylactic acid solution as the oil phase, the oil phase was mixed with an anhydrous ethanol solution containing Maresin-1 at a volume ratio of 8 to 12:1 to obtain an oil phase mixture. Then, an aqueous phase was added to the oil phase mixture, and ultrasonic emulsification was performed to form a primary emulsion. The primary emulsion was mixed with the aqueous phase of the secondary emulsion to form a secondary emulsion. After removing the solvent, the precipitate was collected by centrifugation to obtain polylactic acid nanocarriers loaded with Maresin-1, namely MaR1 nanoparticles.

4. The method for preparing MaR1 nanoparticles according to claim 3, characterized in that, The volume ratio of the oil phase to the anhydrous ethanol solution containing Maresin-1 is 10:

1.

5. The method for preparing MaR1 nanoparticles according to claim 3, characterized in that, The volume ratio of the oil phase to the water phase is 1:5 to 15.

6. The method for preparing MaR1 nanoparticles according to claim 5, characterized in that, The volume ratio of the oil phase to the water phase is 1:

10.

7. The method for preparing MaR1 nanoparticles according to claim 3, characterized in that, The volume ratio of the colostrum to the aqueous phase of the re-emulsion is 1:20 to 40.

8. The method for preparing MaR1 nanoparticles according to claim 3, characterized in that, The concentration of the L-polylactic acid solution is 50 μg / ml to 500 μg / ml.

9. The use of the MaR1 nanoparticles according to claim 1 or 2 in the preparation of a drug for treating diabetic foot ulcers.

10. The application of the MaR1 nanoparticles according to claim 9 in the preparation of a drug for treating diabetic foot ulcers, characterized in that, The drug has the following functions: anti-inflammatory; promotes wound healing; inhibits the formation of pathological scars; and accelerates the regeneration and reconstruction of the epidermal structure.

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