Nano-composite containing antioxidant and siRNA as well as preparation method and application of nano-composite

Abstract

The invention relates to the field of biomedical materials, and discloses a nano-composite containing an antioxidant and siRNA as well as a preparation method and application of the nano-composite. The preparation method comprises the following steps: taking an amino-containing antioxidant and a compound containing a phenylboronic acid structure as raw materials, and obtaining the phenylboronic acid bond modified antioxidant under the catalysis of an onium salt amidation coupling agent; the preparation method comprises the following steps: reacting a phenylboronic acid bond modified antioxidant with a lipid containing a catechol structure in an organic solvent to obtain an antioxidant coupled lipid; the lipid and cationic lipid are mixed and dissolved in a low-boiling-point organic solvent, then a DEPC aqueous solution containing siRNA MMP9 is added, ultrasonic treatment and vacuum concentration are carried out, and the nanocomposite containing the antioxidant and siRNA is obtained. According to the nano-composite prepared by the invention, effective loading of siRNA can be realized only by using two lipids with different functions through electrostatic interaction, and the nano-composite has the advantages of simple preparation method, definite effective components, good biocompatibility and the like.

Description

Technical Field

[0001] The invention relates to the field of biomedical materials, and in particular to a nanocomposite containing an antioxidant and siRNA, and a preparation method and application thereof. Background Art

[0002] Diabetes is a metabolic disease caused by multiple factors. According to the 2021 statistics of the International Diabetes Federation, the number of diabetes patients worldwide has exceeded 500 million, and it is expected that this number will reach 550 million by 2030 and approach 700 million by 2045. China, as a country with a high incidence of diabetes, has more than 140 million patients, and the number of patients is increasing year by year. Chronic wound healing in diabetes is a common complication in diabetic patients, which seriously reduces the quality of life of diabetic patients and may induce systemic diseases. Its clinical treatment has always faced great challenges. Clinically, the main treatments for chronic wound healing in diabetes are debridement, drainage and dressing, but the efficacy is limited. Studies have shown that the wounds of diabetic patients often show highly active inflammatory responses and significant degradation of the extracellular matrix. These factors together make it difficult for diabetic patients to heal their wounds. Excessive inflammatory response is often accompanied by high levels of reactive oxygen species (ROS). Antioxidants can effectively reduce ROS levels, and it is expected to regulate macrophage phenotypes to reduce inflammatory responses. In addition, the high expression of matrix metalloproteinase 9 (MMP9) secreted by macrophages in diabetic wounds is the main cause of accelerated degradation of the extracellular matrix, and it also aggravates the inflammatory response in the wound microenvironment, thereby hindering wound healing. Therefore, downregulating MMP9 expression is expected to enhance the synthesis and remodeling of the extracellular matrix, thereby promoting the healing of diabetic wounds. However, there is still a lack of specific inhibitors of MMP9 in clinical practice, and inhibiting the expression of MMP9 faces great challenges.

[0003] Small interfering RNA (siRNA) drugs can regulate the expression of pathogenic genes at the gene level and silence the target MMP9 without any side effects, showing broad application prospects in the treatment of diabetic wounds. However, siRNA drugs are easily degraded by nucleases, and as hydrophilic macromolecular drugs, they are difficult to be taken up by cells. The effect of siRNA depends largely on its delivery carrier. At present, siRNA carriers include viral vectors and non-viral vectors. Among them, viral vectors have high transfection efficiency, but there are safety issues such as pathogenic mutations and endogenous recombination, which are not conducive to clinical transformation. Non-viral vectors, such as cationic lipids and cationic polymers, have always been a research hotspot for effective loading of siRNA. However, the current delivery system using only cationic carriers faces high hemolysis rate in vivo, and is easily cleared from the body quickly, with potential side effects and other problems. Moreover, there is no report on the combination of antioxidants and siRNA for the treatment of diabetic skin wounds. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and proposes a nanocomplex for co-delivery of antioxidants and siRNA and a preparation method thereof. The nanocomplex is prepared by cationic lipids and antioxidant-modified α-aminophosphonate-derived lipids, and has the ability to simultaneously scavenge reactive oxygen and downregulate MMP9, thereby accelerating the healing of diabetic wounds.

[0005] To achieve the above object, the present invention provides a method for preparing a nanocomposite containing an antioxidant and siRNA, which comprises the following steps:

[0006] 1) Using an antioxidant containing an amino group and a compound containing a phenylboronic acid structure as raw materials, under the catalysis of an onium salt amidation coupling agent, a phenylboronic acid bond-modified antioxidant is obtained;

[0007] 2) reacting the prepared phenylboronic acid bond-modified antioxidant and the catechol-containing α-aminophosphonate-derived lipid in an organic solvent to obtain an antioxidant-coupled α-aminophosphonate-derived lipid;

[0008] 3) The antioxidant-coupled α-aminophosphonate-derived lipid and the commercial cationic lipid are mixed in a mass ratio of 0.125:1 to 8:1, dissolved in a low-boiling point organic solvent, and then a DEPC aqueous solution containing siRNA MMP9 is added according to a molar ratio of the cationic lipid to the amino group / phosphate group (N(DOTAP) / P(siRNA-nt) of 1:1 to 8:1, and the mixture is ultrasonically concentrated under reduced pressure to obtain a nanocomplex containing the antioxidant and siRNA.

[0009] The structural formula of the catechol-containing α-aminophosphonate-derived lipid of the present invention is as follows:

[0010] In the formula, the value of n is preferably 0-10, and more preferably n is 2-6.

[0011] As a further preferred technical solution of the present invention, in step 1), the amino group-containing antioxidant is one of 4-amine-2,2,6,6-tetramethyldiphenylpiperidin (Tempo), spermine, and superoxide dismutase; and / or the compound containing a phenylboronic acid structure is one of 2-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, and 4-carboxyphenylboronic acid, or a combination of two or more thereof; and / or the onium salt amidation coupling agent is one of TBTU, HATU, and HBTU.

[0012] As a further preferred technical solution of the present invention, in step 2), the organic solvent is one or a combination of two or more of N,N-dimethylformamide, methanol and chloroform.

[0013] As a further preferred technical solution of the present invention, in step 3), the commercial cationic lipid is one of DOTAP, DOTMA, and DOSPA; and / or the low boiling point organic solvent is one of chloroform, dichloromethane, ethyl acetate, and methanol, or a combination of two or more thereof.

[0014] As a further preferred technical solution of the present invention, in step 3): the mass ratio of the antioxidant-coupled α-aminophosphonate-derived lipid and the commercial cationic lipid is 0.125:1 to 2:1; and / or the molar ratio of the commercial cationic liposome to the amino group / phosphate group of the siRNA is 3:1.

[0015] As a further preferred technical solution of the present invention, step 1) specifically comprises: dissolving an antioxidant containing an amino group and a compound containing a phenylboronic acid structure in N,N-dimethylformamide, and after complete dissolution, adding an onium salt amidation coupling agent, reacting at 40-45° C. for 24-48 hours, and purifying by column chromatography to obtain an antioxidant modified with a phenylboronic acid bond.

[0016] As a further preferred technical solution of the present invention, step 2) specifically includes: dissolving the antioxidant modified with a phenylboronic acid bond and the α-aminophosphonate-derived lipid containing catechol in methanol at a molar ratio of 1:1, and reacting at 40-45° C. for 6-12 hours to obtain an antioxidant-coupled α-aminophosphonate-derived lipid.

[0017] As a further preferred technical solution of the present invention, step 3) specifically comprises: dissolving the antioxidant-coupled α-aminophosphonate-derived lipid and the commercial cationic lipid in chloroform, mixing them in a mass ratio of 1:1, and then adding a DEPC aqueous solution of siRNA MMP9 according to a molar ratio of amino group / phosphate group of the commercial cationic lipid to siRNA of 3:1, ultrasonically and concentrating under reduced pressure to obtain a nanocomplex containing an antioxidant and siRNA.

[0018] According to another aspect of the present invention, the present invention also provides a nanocomposite, which is prepared by the above method.

[0019] According to another aspect of the present invention, the present invention also provides an application of a nanocomposite in the preparation of a diabetic skin wound repair drug. The nanocomposite can use antioxidants to remove ROS overexpressed in wound tissue at the skin wound site, and the released siMMP9 can downregulate the expression of MMP9 and slow down the degradation of the extracellular matrix, thereby promoting the healing of diabetic skin wounds.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] 1. The nanocomplex prepared by the present invention can effectively load siRNA by using only two lipids with different functions through electrostatic action, and has the advantages of simple preparation method, clear effective ingredients, good biocompatibility, etc.;

[0022] 2. The nanocomplex prepared by the present invention is applied to the repair of diabetic skin wounds. On the one hand, it utilizes the pathological microenvironment to release antioxidants to remove excess reactive oxygen in diabetic wounds and inhibit inflammatory responses; on the other hand, the release of siRNAMMP9 can also downregulate MMP9 expression at the gene level, effectively slowing down the degradation of the extracellular matrix, and achieving combined synergy to promote diabetic wound repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0024] Figure 1 (a) H NMR spectrum and (b) high-resolution mass spectrum of Tempo-pba; (c) H NMR spectrum and (d) high-resolution mass spectrum of Tempo-pba-Lipid.

[0025] Figure 2 (a) When the mass ratio of Lipid-pba-Tempo to DOTAP is 1, the complexation of siRNA at different N(DOTAP) / P(siRNA-nt) ratios; (b) When the N(DOTAP) / P(siRNA-nt) ratio is 3, the complexation of siRNA at different mass ratios of Lipid-pba-Tempo to DOTAP.

[0026] Figure 3 (a) Particle size and (b) Zeta potential of a series of nanocomplexes prepared at different mass ratios of Lipid-pba-Tempo and DOTAP when the N(DOTAP) / P(siRNA-nt) ratio is 3.

[0027] Figure 4 The hemolysis rate of a series of nanocomplexes prepared at different mass ratios of Lipid-pba-Tempo and DOTAP when the N(DOTAP) / P(siRNA-nt) ratio is 3.

[0028] Figure 5 Fluorescence images of a series of nanocomplexes prepared at different mass ratios of Lipid-pba-Tempo and DOTAP to knock down the GFP gene when the N(DOTAP) / P(siRNA-nt) ratio was 3.

[0029] Figure 6When the N(DOTAP) / P(siRNA-nt) ratio is 3, the toxicity of a series of nanocomplexes prepared at different mass ratios of Lipid-pba-Tempo and DOTAP to RAW264.7 cells.

[0030] Figure 7 Figure 2 shows the particle size distribution and transmission electron microscopy image of the optimized nanocomposite.

[0031] Figure 8 The scavenging efficiency of the optimized nanocomplex on different types of reactive oxygen species, including hydrogen peroxide (H2O2), superoxide anion (·O2ˉ), DPPH free radical (DPPH, ·), hydroxyl free radical (·OH), and ABTS free radical (ABTS·).

[0032] Fig. 9 Knockdown of MMP9 gene at (a) mRNA level and (b) protein level after optimization of nanocomplex.

[0033] Fig.10 Image of the effect of the optimized nanocomplex on the wound surface of diabetic mice

[0034] Fig.11 Relative area changes of wounds in diabetic mice treated with optimized nanocomplexes

[0035] Fig.12 Expression of MMP9 gene at mRNA level (a) and protein level (b) in wound tissue of diabetic mice treated with optimized nanocomplex.

[0036] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0037] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0038] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0039] Example 1

[0040] In order to obtain a nanocomposite that can effectively load antioxidants and siRNA and has a high transfection efficiency, this embodiment provides a method for preparing a nanocomposite LNP@T&siRNA containing antioxidants and siRNA, and the specific steps are as follows:

[0041] 1.1 Preparation of phenylboronic acid bond-modified antioxidant (Lipid-pba)

[0042] 0.5 g of 4-amine-2,2,6,6-tetramethyl diphenyl piperate (Tempo), 0.38 g of 4-carboxyphenylboronic acid (pba) and 0.94 g of TBTU were dissolved in 10 mL of N,N-dimethylformamide / pyridine solution (v / v=1:1), stirred and reacted at 45° C. for 24 hours under light-proof conditions, and the organic solvent was removed under reduced pressure. The orange solid phenylboronic acid-modified 4-amine-2,2,6,6-tetramethyl diphenyl piperate Tempo-pba was purified by column chromatography.

[0043] 1.2 Preparation of antioxidant-conjugated lipids (Lipid-pba-Tempo)

[0044] Take 0.32g of the above-mentioned Tempo-pba and 5.99mg of catechol-containing α-aminophosphonate-derived lipid (Lipid), mix them, dissolve them in 0.5mL of methanol, stir them at 45°C overnight under light-proof conditions, and remove the organic solvent under reduced pressure to obtain antioxidant-modified α-aminophosphonate-derived lipid (Tempo-pba-Lipid).

[0045] Among them, the structural formula of catechol-containing α-aminophosphonate-derived lipid (Lipid) is as follows:

[0046]

[0047] 1.3 Preparation of LNP@T&siRNA nanocomplexes

[0048] 0.22 mg of the above-mentioned antioxidant-modified α-aminophosphonate-derived α-lipid Tempo-pba-Lipid and 0.22 mg of the cationic lipid DOTAP were mixed to ensure that the mass ratio of the two was 1:1, dissolved in chloroform, and 500 μL of siRNA DEPC aqueous solution was added respectively, wherein the concentration of siRNA was 5 μM (the corresponding amino / phosphate molar ratio (N(DOTAP) / P(siRNA-nt) was 3, and this ratio is hereinafter referred to as N / P), and then the organic solvent was removed by ultrasound and reduced pressure to obtain the LNP@T&siRNA nanocomplex.

[0049] The H NMR spectra and high-resolution mass spectra of Tempo-pba and Tempo-pba-Lipid prepared in this embodiment are as follows: Figure 1 As shown, the experimental results showed that there were obvious characteristic signal peaks of Tempo-pba and Tempo-pba-Lipid in the H NMR spectrum, and the successful synthesis of Tempo-pba and Tempo-pba-Lipid was further confirmed by high-resolution mass spectrometry.

[0050] Embodiment 2-8

[0051] Based on the preparation method of Example 1, a series of LNP@T&siRNA nanocomplexes were prepared with a mass ratio of Lipid-pba-Tempo and DOTAP of 1:1 and different N(DOTAP) / P(siRNA-nt) ratios. The only difference from Example 1 is that the concentrations of siRNA are 15μM, 7.5μM, 3.75μM, 3.0μM, 2.5μM, 2.14μM, and 1.88μM, respectively, and the corresponding N(DOTAP) / P(siRNA-nt) ratios are 1, 2, 4, 5, 6, 7, and 8, respectively.

[0052] Examples 9-15

[0053] Based on the preparation method of Example 1, 500 μL of DEPC aqueous solution with a siRNA concentration of 5 μM was used, and a series of LNP@T&siRNA nanocomplexes were prepared with different mass ratios of Lipid-pba-Tempo and DOTAP. The only difference from Example 1 is that the amounts of Tempo-pba-Lipid are 0.0275 mg, 0.055 mg, 0.11 mg, 0.44 mg, 0.88 mg, and 1.76 mg, respectively, and the corresponding mass ratios of Lipid-pba-Tempo and DOTAP are 0.125:1, 0.25:1, 0.5:1, 2:1, 4:1, and 8:1.

[0054] The following comprehensive tests were conducted on the nanocomposite samples prepared based on the above Examples 1-15, as follows:

[0055] 1) Characterization of nanocomposites

[0056] The above-obtained series of LNP@T&siRNA nanocomplexes were subjected to electrophoresis at 120V for 30 minutes using 2% agarose gel, gel imaging was performed, and the particle size distribution and Zeta potential of the nanocomplexes were observed using a laser particle size analyzer.

[0057] The prepared nanocomplex LNP@T&siRNA is as follows Figure 2As shown in a, when the mass ratio of Lipid-pba-Tempo to DOTAP is kept at 1 and N(DOTAP) / P(siRNA-nt) is 3, the prepared nanoparticles can completely complex siRNA. Further, by keeping N(DOTAP) / P(siRNA-nt) at 3, by adjusting the mass ratio of antioxidant-coupled lipid Lipid-pba-Tempo and cationic lipid DOTAP, as shown in FIG. Figure 2 As shown in Figure b, the experimental results show that the series of nanoparticles prepared at this time can completely complex siRNA, and the quality of Lipid-pba-Tempo will not affect the complexation effect. In addition, the size and surface potential of the nanocomplex were detected by dynamic light scattering, as shown in Figure 2. Figure 3 As shown in a, when N(DOTAP) / P(siRNA-nt) is kept at 3, the size of the series of nanocomplexes prepared at different mass ratios of Lipid-pba-Tempo and DOTAP is between 50nm and 100nm; Figure 3 As shown in b, the surface potentials of these nanocomposites all maintained a positive charge of +40 mV.

[0058] 2) Hemolysis rate test of nanocomplex LNP@T&siRNA

[0059] When N(DOTAP) / P(siRNA-nt) was 3, the nanocomplex LNP@T&siRNA prepared with different Lipid-pba-Tempo and DOTAP mass ratios was incubated with red blood cell suspension at 4°C for 4 hours, wherein physiological saline and TritonX-100 were used as negative and positive controls, respectively. After the incubation, each sample was centrifuged at 10000rpm for 10 minutes, the supernatant was taken and the absorbance was detected at 450nm using an ELISA reader to calculate the hemolysis rate.

[0060] The hemolysis rate of the series of nanocomplexes LNP@T&siRNA prepared above is as follows Figure 4 As shown, N(DOTAP) / P(siRNA-nt) is 3. When the mass ratio of Lipid-pba-Tempo and DOTAP is less than 2, the obtained series of nanocomplexes maintain extremely low hemolysis rates, all below 5%, indicating that the nanocomplexes have good biocompatibility.

[0061] 3) Nanocomplex LNP@T&siRNA knockdown GFP reporter gene test

[0062] GFP-labeled RAW264.7 cells were seeded in 24-well plates, with 20,000 cells per well. A series of nanocomplexes LNP@T&siRNA prepared with different Lipid-pba-Tempo and DOTAP mass ratios at N(DOTAP) / P(siRNA-nt) of 3 were transfected into GFP-RAW364.7 cells under serum-free conditions for 6 hours, then replaced with complete medium and cultured for 48 hours. The expression of GFP in RAW364.7 was observed by biofluorescence microscopy. The LipoRNAiMax group was used as a positive control.

[0063] The knockout effect of the prepared series of nanocomplexes LNP@T&siRNA delivery siRNA GFP gene on the reporter gene is as follows Figure 5 As shown, untreated GFP-RAW364.7 cells exhibited bright green fluorescence, while the green fluorescence of the series of nanocomplexes LNP@T&siRNA was significantly weakened, just like the LipoRNAiMax group, and was almost invisible.

[0064] 4) Nanocomplex LNP@T&siRNA cytotoxicity test

[0065] RAW264.7 cells were seeded in 96-well plates, with 5000 cells per well. When N(DOTAP) / P(siRNA-nt) was 3, a series of nanocomplexes LNP@T&siRNA prepared with different Lipid-pba-Tempo and DOTAP mass ratios were added to the 96-well plates and incubated for 24 hours, and then the cell viability was detected using CCK8 reagent.

[0066] The cytotoxicity of the prepared series of nanocomplexes LNP@T&siRNA is as follows Figure 6 As shown in the figure, with the increase of the mass ratio of Lipid and DOTAP, the cell viability gradually decreased, especially when the mass ratio was 2, the cell viability of RAW264.7 cells was less than 80%. Therefore, we selected N(DOTAP) / P(siRNA-nt) as 3 and the mass ratio of Lipid-pba-Temp and DOTAP as 1, which had the best effect as the optimized nanocomplex LNP@T&siRNA.

[0067] In summary, the performance tests of the series of complexes in 1)-4) above indicate that the mass ratio of Lipid-pba-Tempo to DOTAP in the system of the present invention is between 0.125:1 and 8:1, and the N / P ratio is between 1:1 and 8:1, which can meet the requirements of the invention.

[0068] The optimized nanocomposite particles LNP@T&siRNA (the mass ratio of Lipid-pba-Tempo and DOTAP is 1, and the ratio of N(DOTAP) / P(siRNA-nt) is 3) were tested as follows:

[0069] 1) Size distribution and morphology of LNP@T&siRNA nanocomplex

[0070] The size distribution of the nanoparticles was detected by dynamic light scattering; the nanoparticles were dropped onto a copper grid, and after drying, the morphology of the nanoparticles was observed using a transmission electron microscope.

[0071] The morphology of LNP@T&siRNA obtained by the present invention is as follows: Figure 7 As shown, the size distribution of the nanocomplex is about 60 nm, and the TEM experimental results show that LNP@T&siRNA has a regular spherical morphology.

[0072] 2) ROS scavenging ability of LNP@T&siRNA nanocomplex

[0073] The superoxide anion kit, DPPH free radical kit, hydroxyl free radical kit and ABTS free radical kit were used to detect the scavenging effect of LNP@T&siRNA nanocomplex on reactive oxygen species at different Tempo concentrations.

[0074] The present invention optimizes the obtained nanocomplex LNP@T&siRNA to remove different types of reactive oxygen species. Figure 8 As shown, LNP@T&siRNA has a broad spectrum of reactive oxygen scavenging ability, including H2O2, ·O2ˉ, DPPH·, ·OH and ABTS· free radicals, and with the increase of Tempo concentration, the reactive oxygen scavenging ability gradually increases, and some types of reactive oxygen can be 100% cleared.

[0075] 3) In vitro transfection effect of nanocomplex LNP@T&siMMP9

[0076] RAW264.7 cells (2.0×105 cells / well) were cultured in a 24-well plate overnight, and then pre-stimulated with LPS (1.0 μg / mL) for 12 hours, followed by pre-treatment with serum-free medium containing the nanocomplex LNP@T&siMMP9 for 6 hours, where the siMMP9 concentration was 25, 50, and 100 nM. Among them, LipoRNAiMax was used as a positive control group and siNC was used as a negative control group. After the co-incubation, fresh complete medium was replaced and cultured for another 24 hours, and the cells were collected. The expression of the MMP9 gene at the mRNA and protein levels was detected by q-PCR and Western Blot.

[0077] The knockout results of the optimized nanocomplex LNP@T&siRNA nanocomplex on the MMP9 gene in RAW264.7 cells are as follows: Fig. 9 As shown in the figure, RT-qPCR and Western Blot experiments showed that siMMP9 delivered by LipoRNAiMax can significantly knock down the MMP9 gene at the mRNA and protein levels; using the nanocomplex LNP@T&siMMP9 developed by us, when the siRNA concentration is 100nM, the MMP9 gene can be knocked down to about 50% at the mRNA and protein levels. These experimental results show that the nanocomplex LNP@T&siMMP9 can effectively inhibit the expression of MMP9 at the in vitro level.

[0078] 4) Nanocomplex LNP@T&siMMP9 promotes wound repair in diabetic mice

[0079] ICR mice aged 6 to 8 weeks were selected and injected with streptozotocin (STZ) at a dose of 100 mg / kg each time for 7 consecutive days. When the fasting blood glucose value was ≥16.7 mmol / L, the diabetic model was considered to be successfully established. A skin perforator with a diameter of 8 mm was used to establish a full skin injury model on the back of diabetic mice. After the diabetic chronic wound healing model was successfully established, the mice were randomly divided into 5 groups, including PBS group, Free siMMP9 group, LNP@T group, LNP@T&siNC group and LNP@T&siMMP9 group; 50 μL of various preparations were dripped into the wound site, and the drug was administered every other day. The wound healing images were collected and the wound area was calculated. After the treatment, the skin wounds of the mice were collected, and the expression of MMP9 gene in the wound tissue was analyzed by RT-qPCR and Western Blot technology.

[0080] The therapeutic effect of the optimized nanocomplex LNP@T&siRNA on the wound of diabetic mice is as follows Fig.10 and Fig.11 As shown in Figure 2, the skin wound healing effect of mice in the LNP@T&siMMP9 group was significantly better than that in other control groups. Fig.12 As shown, the skin wounds of diabetic mice were collected, and RT-qPCR and Western Blot analysis showed that the MMP9 gene in the skin tissue of mice in the LNP@T&siMMP9 group was significantly downregulated both at the mRNA level and at the protein level.

[0081] Although specific embodiments of the present invention are described above, those skilled in the art should understand that these are merely examples and that various changes or modifications may be made to the embodiments without departing from the principles and essence of the present invention. The scope of protection of the present invention is limited only by the appended claims.

Claims

1. A method for preparing a nanocomposite containing an antioxidant and siRNA, characterized in that: The following steps are involved: 1) Using an antioxidant containing an amino group and a compound containing a phenylboronic acid structure as raw materials, under the catalysis of an onium salt amidation coupling agent, a phenylboronic acid bond-modified antioxidant is obtained; 2) reacting the prepared phenylboronic acid bond-modified antioxidant and the catechol-containing α-aminophosphonate-derived lipid in an organic solvent to obtain an antioxidant-coupled α-aminophosphonate-derived lipid; 3) The antioxidant-coupled α-aminophosphonate-derived lipid and the commercial cationic lipid are mixed at a mass ratio of 0.125:1 to 8:1, dissolved in a low-boiling point organic solvent, and then a DEPC aqueous solution containing siRNA MMP9 is added according to a molar ratio of the cationic lipid to the amino group / phosphate group in the siRNA of 1:1 to 8:1, and the mixture is ultrasonically concentrated under reduced pressure to obtain a nanocomplex containing the antioxidant and the siRNA.

2. The method for preparing a nanocomposite containing an antioxidant and siRNA according to claim 1, characterized in that: In step 1), the amino group-containing antioxidant is one of 4-amine-2,2,6,6-tetramethyldiphenylpiperidin, spermine, and superoxide dismutase; and / or the compound containing a phenylboronic acid structure is one of 2-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, and 4-carboxyphenylboronic acid, or a combination of two or more thereof; and / or the onium salt amidation coupling agent is one of TBTU, HATU, and HBTU.

3. The method for preparing a nanocomposite containing an antioxidant and siRNA according to claim 1, characterized in that: In step 2), the organic solvent is one or a combination of two or more of N,N-dimethylformamide, methanol and chloroform.

4. The method for preparing a nanocomposite containing an antioxidant and siRNA according to claim 1, characterized in that: In step 3), the commercial cationic lipid is one of DOTAP, DOTMA and DOSPA; and / or the low boiling point organic solvent is one of chloroform, dichloromethane, ethyl acetate and methanol, or a combination of two or more thereof.

5. The method for preparing a nanocomposite containing an antioxidant and siRNA according to claim 1, characterized in that: In step 3), the mass ratio of the antioxidant-coupled α-aminophosphonate-derived lipid to the commercial cationic lipid is 0.125:1 to 2:1; and / or the N / P ratio of the commercial cationic liposome to siRNA is 3:

1.

6. The method for preparing a nanocomposite containing an antioxidant and siRNA according to any one of claims 1 to 5, characterized in that: Step 1) specifically comprises: dissolving an antioxidant containing an amino group and a compound containing a phenylboronic acid structure in N,N-dimethylformamide, adding an onium salt amidation coupling agent after complete dissolution, reacting at 40-45° C. for 24-48 hours, and purifying by column chromatography to obtain an antioxidant modified with a phenylboronic acid bond.

7. The method for preparing a nanocomposite containing an antioxidant and siRNA according to any one of claims 1 to 5, characterized in that: Step 2) specifically comprises: dissolving the antioxidant modified with phenylboronic acid bond and the catechol-containing α-aminophosphonate-derived lipid in methanol at a molar ratio of 1:1, and reacting at 40-45° C. for 6-12 hours to obtain the antioxidant-coupled lipid.

8. The method for preparing a nanocomposite containing an antioxidant and siRNA according to any one of claims 1 to 5, characterized in that: Step 3) specifically includes: dissolving the antioxidant-coupled α-aminophosphonate-derived lipid and the commercial cationic lipid in chloroform, mixing them in a mass ratio of 1:1, then adding the DEPC aqueous solution of siRNA MMP9 according to the molar ratio of the commercial cationic lipid to the amino group / phosphate group of siRNA of 3:1, sonicating, and concentrating under reduced pressure to obtain a nanocomplex containing antioxidants and siRNA.

9. A nanocomposite, characterized in that: The method is prepared by any one of claims 1 to 8.

10. Use of the nanocomposite according to claim 9 in preparing a drug for repairing diabetic skin wounds.

Images