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 present application relates to the field of biomedical materials, in particular to a nano-complex containing an antioxidant and siRNA, and a preparation method and application thereof. BACKGROUND

[0002] Diabetes is a metabolic disease caused by multiple factors. Chronic wound healing in diabetes is a common complication in diabetic patients, which seriously reduces the quality of life of diabetic patients and can induce systemic diseases, and its clinical treatment has always been a great challenge. Clinically, the treatment of chronic wound healing in diabetes mainly focuses on debridement, drainage and dressing, but the effect is limited. Studies have shown that the wound of diabetic patients often presents a high activity of inflammatory response and a significant degradation of extracellular matrix, which together lead to the difficulty of wound healing in diabetic patients. Excessive inflammatory response is often accompanied by high levels of reactive oxygen species (ROS), which can be effectively reduced by antioxidants, and is expected to regulate macrophage phenotype to reduce inflammatory response. In addition, the high expression of matrix metalloproteinase 9 (MMP9) secreted by macrophages at the site of diabetic wound is the main reason for the accelerated degradation of extracellular matrix, and it also aggravates the inflammatory response in the wound microenvironment, thereby hindering wound healing. Therefore, down-regulating the expression of MMP9 is expected to enhance the synthesis and remodeling of extracellular matrix, thereby promoting the healing of diabetic wounds. However, there is still a lack of specific inhibitors of MMP9 in clinical practice, and it is a great challenge to inhibit the expression of MMP9.

[0003] Small interfering RNA (siRNA) drugs can regulate the expression of pathogenic genes at the gene level, silence target MMP9 without any side effects, and show 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, and the effect of siRNA is greatly dependent on its delivery carrier. The current siRNA carriers include viral vectors and non-viral vectors, among which the transfection efficiency of viral vectors is higher, but there are safety problems such as pathogenic mutation and endogenous recombination, which are not conducive to clinical transformation. Non-viral carriers, such as cationic lipids and cationic polymers, have always been a research hotspot for effective siRNA loading. However, the current delivery system using single cationic carrier in vivo faces high hemolysis rate, and is easily cleared from the body, which has potential side effects. Moreover, there is no report on the use of antioxidants and siRNA for the treatment of diabetic skin wounds. SUMMARY

[0004] The present application aims to overcome the shortcomings and deficiencies of the prior art, and provides a co-delivery nanocomposite containing an antioxidant and siRNA and a preparation method thereof, which is prepared from a cationic lipid and an antioxidant-modified alpha-amino phosphonate derivative lipid, so as to solve the problems of high hemolysis rate, rapid clearance from the body, potential side effects and the like of the existing lipid carrier delivery system for siRNA in the body.

[0005] To achieve the above-mentioned object, the present application provides a preparation method of a nanocomposite containing an antioxidant and siRNA, comprising the following steps:

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

[0007] 2) Reacting the prepared antioxidant modified by a phenylboronic acid bond and an alpha-amino phosphonate derivative lipid containing catechol in an organic solvent to obtain an antioxidant-coupled alpha-amino phosphonate derivative lipid;

[0008] 3) Mixing the antioxidant-coupled alpha-amino phosphonate derivative lipid and a commercial cationic lipid at a mass ratio of 0.125:1~8:1, dissolving them in a low-boiling organic solvent, and then adding a DEPC aqueous solution containing siRNA MMP9 according to the molar ratio of cationic lipid to amino group / phosphate group (N(DOTAP) / P(siRNA-nt) of 1:1~8:1, ultrasonic treatment, and vacuum concentration to obtain a nanocomposite containing an antioxidant and siRNA.

[0009] The structural formula of the alpha-amino phosphonate derivative lipid containing catechol in the present application 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 application, in step 1), the antioxidant containing an amino group is one of 4-amine-2,2,6,6-tetramethylpiperidyl (Tempo), spermine, and superoxide dismutase; and / or, the compound containing a phenylboronic acid structure is one or a combination of two or more of 2-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, and 4-carboxyphenylboronic acid; and / or, the onium salt amide coupling agent is one of TBTU, HATU, and HBTU.

[0012] As a further preferred technical solution of the present application, 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 application, in step 3), the commercial cationic lipid is one of DOTAP, DOTMA and DOSPA; and / or, the low-boiling organic solvent is one or a combination of two or more of chloroform, dichloromethane, ethyl acetate and methanol.

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

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

[0016] As a further preferred technical solution of the present application, step 2) specifically comprises: dissolving the antioxidant modified by phenylboronic acid bond and the α-amino phosphonate derivative lipid containing catechol in methanol according to a molar ratio of 1:1, and reacting at 40-45 °C for 6-12 hours to obtain the antioxidant-coupled α-amino phosphonate derivative lipid.

[0017] As a further preferred technical solution of the present application, step 3) specifically comprises: dissolving the antioxidant-coupled α-amino phosphonate derivative lipid and the commercial cationic lipid in chloroform, mixing according to a mass ratio of 1:1, then adding the DEPC aqueous solution of siRNA MMP9 (abbreviated as siMMP9) according to the molar ratio of the commercial cationic lipid and the amino / phosphate of siRNA of 3:1, ultrasonicating and concentrating under reduced pressure to obtain the nanocomposite containing the antioxidant and siRNA MMP9.

[0018] Preferably, the antisense strand nucleotide sequence of siRNA MMP9 is 5'-GGUGUGCGACCACAUCGAATT-3', 5'-GCACUGGGCUUAGAUCAUUTT-3', 5'-AAUGAUCUAAGCCCAGUGCTT-3'.

[0019] According to another aspect of the present application, the present application also provides a nanocomposite prepared by the above method.

[0020] According to another aspect of the present invention, the present invention also provides the application of a nanocomposite in the preparation of a drug for repairing diabetic skin wounds. This nanocomposite, at the skin wound site, can, on the one hand, utilize antioxidants to scavenge overexpressed ROS in the wound tissue, and on the other hand, the released siMMP9 can downregulate MMP9 expression, slowing down extracellular matrix degradation, thereby promoting the healing of diabetic skin wounds.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] 1. The nanocomposite prepared by this invention can effectively load siRNA using only two lipids with different functions through electrostatic interaction. The effective loading capacity is not affected by the nucleotide sequence of siRNA. It also has the advantages of simple preparation method, clear effective components, and good biocompatibility.

[0023] 2. The nanocomposite prepared in this invention has broad applicability for the effective loading of siRNA, and can be extended to other nucleotide sequences besides siRNA targeting GFP and MMP9. Moreover, it can perfectly solve the problems of high hemolysis rate and rapid clearance from the body faced by lipid carrier delivery systems in vivo.

[0024] 3. The nanocomposite prepared in this 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 species in diabetic wounds and inhibit inflammatory responses. On the other hand, the release of siRNA MMP9 can also downregulate MMP9 expression at the gene level, effectively slowing down the degradation of the extracellular matrix, thereby achieving a synergistic effect to promote the repair of diabetic wounds. Attached Figure Description

[0025] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0026] Figure 1 (a) 1H NMR spectrum and (b) high-resolution mass spectrum of Tempo-pba; (c) 1H NMR spectrum and (d) high-resolution mass spectrum of Tempo-pba-Lipid.

[0027] Figure 2 (a) When the mass ratio of Lipid-pba-Tempo to DOTAP is 1, the complexation of siRNA under different N(DOTAP) / P(siRNA-nt) ratios; (b) When the N(DOTAP) / P(siRNA-nt) ratio is 3, the complexation of siRNA under different mass ratios of Lipid-pba-Tempo to DOTAP.

[0028] Figure 3(a) Particle size and (b) Zeta potential of a series of nanocomposites prepared with different mass ratios of Lipid-pba-Tempo and DOTAP when the N(DOTAP) / P(siRNA-nt) ratio is 3.

[0029] Figure 4 The hemolysis rate of a series of nanocomposites prepared with different mass ratios of Lipid-pba-Tempo and DOTAP when the N(DOTAP) / P(siRNA-nt) ratio is 3.

[0030] Figure 5 Fluorescence images of a series of nanocomposites prepared with different mass ratios of Lipid-pba-Tempo and DOTAP to knock down the GFP gene when the N(DOTAP) / P(siRNA-nt) ratio is 3.

[0031] Figure 6 The toxicity of a series of nanocomposites prepared with different mass ratios of Lipid-pba-Tempo and DOTAP to RAW264.7 cells when the N(DOTAP) / P(siRNA-nt) ratio is 3.

[0032] Figure 7 The optimized nanocomposite particle size distribution and transmission electron microscopy image.

[0033] Figure 8 The optimized nanocomposite was designed to scavenge different types of reactive oxygen species, including hydrogen peroxide (H2O2), superoxide anion (•O2ˉ), DPPH radical (DPPH•), hydroxyl radical (•OH), and ABTS radical (ABTS•).

[0034] Figure 9 The optimized nanocomposite was used to knock down the MMP9 gene at (a) mRNA level and (b) protein level.

[0035] Figure 10 Images showing the effect of the optimized nanocomposite on the wounds of diabetic mice.

[0036] Figure 11 The relative area change of wounds in diabetic mice treated with the optimized nanocomposite.

[0037] Figure 12 Expression of the MMP9 gene at the mRNA level (a) and protein level (b) in wound tissue of diabetic mice treated with the optimized nanocomposite.

[0038] The objectives, features, and advantages of this invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0039] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0040] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0041] Terminology Explanation: siRNA MMP9 refers to the use of small interfering RNA (siRNA) technology to target and silence the expression of the matrix metalloproteinase-9 (MMP9) gene.

[0042] The antisense strand sequence of siRNA MMP9 that can be used in this invention can be: 5'-GGUGUGCGACCACAUCGAATT-3', 5'-AAUGAUCUAAGCCCAGUGCTT-3', 5'-GCACUGGGCUUAGAUCAUUTT-3', 5'-GCAUGAACUCCUCAAGUAA-3', 5'-CCUGAACUGAAGCUCAUGA-3', 5'-AAGCUCAUGAACUGAAGCU-3', etc.

[0043] Example 1

[0044] To obtain a nanocomposite capable of effectively loading antioxidants and siRNA, and exhibiting high transfection efficiency, this embodiment provides a method for preparing the LNP@T&siRNA nanocomposite containing antioxidants and siRNA. The specific steps are as follows:

[0045] 1.1 Preparation of phenylboronic acid bond-modified antioxidants (Lipid-pba)

[0046] 0.5 g of 4-amine-2,2,6,6-tetramethyldiphenylpiperate (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). The reaction was carried out at 45°C for 24 hours under light-protected conditions with stirring. The organic solvent was removed under reduced pressure, and the 4-amine-2,2,6,6-tetramethyldiphenylpiperate (Tempo-pba) was purified by column chromatography to obtain an orange solid modified with phenylboronic acid.

[0047] 1.2 Preparation of antioxidant-coupled lipids (Lipid-pba-Tempo)

[0048] Take 0.32 g of the above Tempo-pba and 5.99 mg of α-aminophosphonate-derived lipid (Lipid) containing catechol, mix them, dissolve them in 0.5 mL of methanol, stir overnight at 45°C under light protection, remove the organic solvent under reduced pressure, and obtain antioxidant-modified α-aminophosphonate-derived lipid (Tempo-pba-Lipid).

[0049] The structural formula of the α-aminophosphonate-derived lipid (Lipid) containing catechol is shown below:

[0050] .

[0051] 1.3 Preparation of LNP@T&siRNA nanocomposites

[0052] Take 0.22 mg of the antioxidant-modified α-aminophosphonate-derived α-lipid Tempo-pba-Lipid and 0.22 mg of cationic lipid DOTAP, ensuring a mass ratio of 1:1, dissolve in chloroform, add 500 μL of DEPC aqueous solution of siRNA MMP9 (antisense nucleotide sequence 5'-GGUGUGCGACCACAUCGAATT-3'), where the concentration of siRNA is 5 μM (corresponding to an amino / phosphate molar ratio (N(DOTAP) / P(siRNA-nt) is 3, hereinafter referred to as N / P), and then remove the organic solvent by sonication and reduced pressure to obtain the LNP@T&siRNA nanocomposite.

[0053] The 1H NMR 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 indicate that there are obvious characteristic signal peaks of Tempo-pba and Tempo-pba-Lipid in the 1H NMR spectrum, and the successful synthesis of Tempo-pba and Tempo-pba-Lipid was further confirmed by high-resolution mass spectrometry.

[0054] Examples 2-8

[0055] Based on the preparation method of Example 1, a series of LNP@T&siRNA nanocomposites were prepared with a mass ratio of Lipid-pba-Tempo to DOTAP of 1:1 and different N(DOTAP) / P(siRNA-nt) ratios. The only difference between these and Example 1 was that the concentrations of siRNA were 15 μM, 7.5 μM, 3.75 μM, 3.0 μM, 2.5 μM, 2.14 μM, and 1.88 μM, and the corresponding N(DOTAP) / P(siRNA-nt) ratios were 1, 2, 4, 5, 6, 7, and 8, respectively.

[0056] Examples 9-15

[0057] Based on the preparation method of Example 1, 500 μL of DEPC aqueous solution with a concentration of 5 μM siRNA was used to prepare a series of LNP@T&siRNA nanocomposites with different mass ratios of Lipid-pba-Tempo and DOTAP. The only difference from Example 1 was that the amount of Tempo-pba-Lipid used was 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 were 0.125:1, 0.25:1, 0.5:1, 2:1, 4:1, and 8:1.

[0058] The following is a comprehensive test of the nanocomposite samples prepared in Examples 1-15 above, as detailed below:

[0059] 1) Characterization of nanocomposites

[0060] The series of LNP@T&siRNA nanocomposites obtained above were subjected to gel electrophoresis at 120 V for 30 minutes using 2% agarose gel, and the particle size distribution and zeta potential of the nanocomposites were observed using a laser particle size analyzer.

[0061] The series of LNP@T&siRNA nanocomposites prepared above are as follows: Figure 2 As shown in Figure a, it can be seen that when the mass ratio of Lipid-pba-Tempo to DOTAP is kept at 1 and the N(DOTAP) / P(siRNA-nt) ratio is 3, the prepared nanoparticles can completely complex siRNA. Furthermore, while keeping the N(DOTAP) / P(siRNA-nt) ratio at 3, the mass ratio of the antioxidant-coupled lipid Lipid-pba-Tempo and the cationic lipid DOTAP can be adjusted, such as... Figure 2As shown in Figure b, the experimental results indicate that the series of nanoparticles prepared at this time can completely complex siRNA, and the quality of Lipid-pba-Tempo does not affect the complexation effect. Furthermore, the size and surface potential of the nanocomposite were detected by dynamic light scattering, such as... Figure 3 As shown in Figure a, while maintaining N(DOTAP) / P(siRNA-nt) at 3, the sizes of a series of nanocomposites prepared with different mass ratios of Lipid-pba-Tempo and DOTAP ranged from 50 nm to 100 nm; Figure 3 As shown in Figure b, the surface potential of these nanocomposites remains positively charged, at +40 mV.

[0062] Furthermore, based on Examples 1-5 above, only the siRNA sequence was changed to other siRNA MMP9 sequences, such as 5'-AAUGAUCUAAGCCCAGUGCTT-3', 5'-GCACUGGGCUUAGAUCAUUTT-3', 5'-GCAUGAACUCCUCAAGUAA-3', 5'-CCUGAACUGAAGCUCAUGA-3', and 5'-AAGCUCAUGAACUGAAGCU-3'. The complexation of siRNA, particle size, and zeta potential distribution in the LNP@T&siRNA nanocomposites prepared by changing the siRNA were the same as in Examples 1-15. The test results show that when the mass ratio of antioxidant-coupled α-aminophosphonate-derived lipids and commercially available cationic lipids is 0.125:1 to 8:1, and when the molar ratio of cationic lipids to amino / phosphate groups in siRNA is 1:1 to 8:1, nanocomposites containing antioxidants and siRNA can be successfully prepared. Furthermore, the loading capacity of the nanolipid delivery system formed by antioxidant-coupled α-aminophosphonate-derived lipids and commercially available cationic lipids in this invention for siRNA is independent of the siRNA sequence.

[0063] 2) Hemolysis rate test of LNP@T&siRNA nanocomposite

[0064] When the N(DOTAP) / P(siRNA-nt) ratio was 3, the nanocomplex LNP@T&siRNA prepared with different Lipid-pba-Tempo and DOTAP mass ratios was co-incubated with erythrocyte suspension at 4°C for 4 hours. Physiological saline and Triton X-100 were used as negative and positive controls, respectively. After incubation, each sample was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. The absorbance was measured at 450 nm using a microplate reader, and the hemolysis rate was calculated.

[0065] The hemolysis rate of the series of LNP@T&siRNA nanocomposites prepared above is as follows: Figure 4 As shown, when the N(DOTAP) / P(siRNA-nt) ratio is 3 and the mass ratio of Lipid-pba-Tempo to DOTAP is less than 2, the obtained series of nanocomposites maintain extremely low hemolysis rates, all below 5%, indicating that the nanocomposites have good biocompatibility.

[0066] Furthermore, referring to the preparation methods of Examples 1-15 above, several nanocomplexes LNP@T&siRNA containing different siRNA sequences were prepared by simply changing the nucleotide sequence of the siRNA, such as 5'-AAUGAUCUAAGCCCAGUGCTT-3', 5'-GCACUGGGCUUAGAUCAUUTT-3', 5'-GCAUGAACUCCUCAAGUAA-3', 5'-CCUGAACUGAAGCUCAUGA-3', 5'-AAGCUCAUGAACUGAAGCU-3', etc. The hemolytic test results were as follows: N(DOTAP) / P(siRNA-nt) was 3. When the mass ratio of Lipid-pba-Tempo to DOTAP was less than 2, the hemolysis rate was less than 5%, maintaining an extremely low level. Experimental results show that hemolysis is independent of the siRNA sequence. The nanolipid delivery system formed by antioxidant-coupled α-aminophosphonate-derived lipids and commercially available cationic lipids in this invention can effectively solve the problem of high hemolysis rate and rapid clearance from the body faced by the target lipid delivery system in vivo.

[0067] 3) Nanocomposite LNP@T & siRNA knockdown of GFP reporter gene assay

[0068] GFP-labeled RAW264.7 cells were seeded in 24-well plates at a density of 20,000 cells per well. Based on the preparation method in Example 1, a series of nanocomplexes LNP@T&siRNA (where the antisense nucleotide sequence of siRNA GFP is 5'-CAAGCUGACCCUGAAGUUCtt-3') were prepared with an N(DOTAP) / P(siRNA-nt) ratio of 3 using different Lipid-pba-Tempo and DOTAP mass ratios. These nanocomplexes were transfected into GFP-RAW364.7 cells for 6 hours under serum-free conditions, followed by replacement with complete culture medium and culturing for another 48 hours. The expression of green fluorescent protein (GFP) in RAW364.7 cells was observed using a biofluorescence microscope. The LipoRNAiMax group served as a positive control.

[0069] The above-prepared series of nanocomposites, LNP@T&siRNA, delivered siRNA-GFP gene, and their knockout effect on the reporter gene are as follows: Figure 5 As shown, untreated GFP-RAW364.7 cells exhibited bright green fluorescence, while the green fluorescence of the LNP@T&siRNA nanocomplex was significantly reduced, almost to the point of being invisible, similar to the LipoRNAiMax group.

[0070] 4) Cytotoxicity test of LNP@T&siRNA nanocomposite

[0071] RAW264.7 cells were seeded in 96-well plates at a density of 5000 cells per well. Based on the preparation method in Example 1, a series of nanocomplexes LNP@T&siRNA were prepared with different Lipid-pba-Tempo and DOTAP mass ratios at an N(DOTAP) / P(siRNA-nt) ratio of 3. These nanocomplexes were then added to the 96-well plates and incubated for 24 hours. Cell viability was then assessed using CCK8 reagent. The antisense nucleotide sequence of siRNA MMP9 was 5'-GGUGUGCGACCACAUCGAATT-3'.

[0072] The cytotoxicity of the series of LNP@T&siRNA nanocomposites prepared above is as follows: Figure 6 As shown, cell viability gradually decreased with increasing Lipid to DOTAP mass ratio, especially at a mass ratio of 2, where RAW264.7 cell viability fell below 80%. Therefore, we selected an N(DOTAP) / P(siRNA-nt) ratio of 3 and a Lipid-pba-Temp to DOTAP mass ratio of 1, which yielded the best results, as the optimized nanocomplex LNP@T&siRNA.

[0073] In summary, the performance tests of the series of complexes in 1)-4) above demonstrate that the mass ratio of Lipid-pba-Tempo to DOTAP in the system of this invention is between 0.125:1 and 8:1, and the N / P ratio is between 1:1 and 8:1, which meets the requirements of the invention.

[0074] The following tests were performed on the optimized nanocomposite LNP@T&siRNA (Lipid-pba-Tempo and DOTAP mass ratio of 1, N(DOTAP) / P(siRNA-nt) ratio of 3), wherein the antisense strand nucleotide sequence of siRNA MMP9 is 5'-GGUGUGCGACCACAUCGAATT-3':

[0075] 1) Size distribution and morphology of the LNP@T&siRNA nanocomposite

[0076] The size distribution of the nanoparticles was detected by dynamic light scattering; the nanoparticles were then dropped onto a copper grid and, after drying, their morphology was observed using a transmission electron microscope.

[0077] The morphology of the optimized LNP@T&siRNA obtained in this invention is as follows: Figure 7 As shown, the size distribution of the nanocomposite is approximately 60 nm, and TEM results indicate that LNP@T&siRNA has a regular spherical morphology.

[0078] 2) Reactive oxygen species scavenging ability of the LNP@T&siRNA nanocomposite

[0079] The scavenging effect of LNP@T&siRNA nanocomplex on reactive oxygen species at different Tempo concentrations was detected using a superoxide anion assay kit, a DPPH radical assay kit, a hydroxyl radical assay kit, and an ABTS radical assay kit.

[0080] The optimized nanocomposite LNP@T&siRNA obtained in this invention exhibits the following scavenging abilities against different types of reactive oxygen species: Figure 8 As shown, LNP@T&siRNA has a broad spectrum of reactive oxygen species (ROS) scavenging ability, including H2O2, •O2ˉ, DPPH•, •OH and ABTS• free radicals. Moreover, the ROS scavenging ability gradually increases with the increase of Tempo concentration, and it can achieve 100% scavenging of some types of ROS.

[0081] 3) In vitro transfection effect of LNP@T&siMMP9 nanocomposite

[0082] RAW264.7 cells (2.0 × 10⁵ cells / well) were cultured overnight in 24-well plates, then pre-stimulated with LPS (1.0 μg / mL) for 12 hours, followed by pretreatment with serum-free medium containing the LNP@T&siMMP9 nanocomplex at concentrations of 25, 50, and 100 nM for 6 hours. LipoRNAiMax served as a positive control, and siNC as a negative control. After co-incubation, the medium was replaced with fresh complete medium and cultured for another 24 hours. Cells were then harvested, and q-PCR and Western blotting were used to detect the expression of the MMP9 gene at both mRNA and protein levels.

[0083] The knockout results of the optimized LNP@T&siRNA nanocomposite in RAW264.7 cells on the MMP9 gene are as follows: Figure 9As shown in the results, RT-qPCR and Western Blot experiments demonstrated that siMMP9 delivery using LipoRNAiMax significantly knocked down the MMP9 gene at both the mRNA and protein levels. Furthermore, using our developed nanocomposite LNP@T&siMMP9 at a siRNA concentration of 100 nM, the MMP9 gene was knocked down to approximately 50% at both the mRNA and protein levels. These experimental results indicate that the LNP@T&siMMP9 nanocomposite can effectively inhibit MMP9 expression in vitro.

[0084] 4) The LNP@T&siMMP9 nanocomposite promotes wound repair in diabetic mice.

[0085] ICR mice aged 6-8 weeks were selected and injected with streptozotocin (STZ) at a dose of 100 mg / kg for 7 consecutive days. A diabetes model was considered successfully established when the fasting blood glucose level was ≥16.7 mmol / L. A full-skin lesion model was established on the back of the diabetic mice using an 8 mm diameter skin perforator. After the diabetic chronic healing wound model was successfully established, the mice were randomly divided into 5 groups: PBS group, Free siMMP9 group, LNP@T group, LNP@T&siNC group, and LNP@T&siMMP9 group. 50 μL of each formulation was applied to the wound site every other day, and images of wound healing were collected to calculate the wound area. After treatment, the mouse skin wounds were collected, and the expression of the MMP9 gene in the wound tissue was analyzed using RT-qPCR and Western Blot techniques.

[0086] The therapeutic effect of the optimized nanocomposite LNP@T&siRNA on wounds in diabetic mice is as follows: Figure 10 and Figure 11 As shown, the LNP@T&siMMP9 group mice exhibited significantly better skin wound healing than other control groups. Furthermore, as... Figure 12 As shown, skin wounds were collected from diabetic mice, and RT-qPCR and Western Blot analysis revealed that the MMP9 gene was significantly downregulated in the skin tissue of mice in the LNP@T&siMMP9 group, both at the mRNA and protein levels.

[0087] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. The scope of protection of the present invention is defined only by the appended claims.

Claims

1. A method for preparing a nanocomplex containing an antioxidant and an siRNA, characterized by, The method comprises the following steps: 1) using an amino-containing antioxidant and a compound containing a phenylboronic acid structure as raw materials, and obtaining an antioxidant with a phenylboronic acid bond under the catalysis of an onium salt amide coupling agent; 2) reacting the prepared antioxidant with a phenylboronic acid bond and an α-aminophosphonate derivative lipid containing catechol in an organic solvent to obtain an antioxidant-coupled α-aminophosphonate derivative lipid; 3) mixing the antioxidant-coupled α-aminophosphonate derivative lipid and a commercial cationic lipid at a mass ratio of 0.125:1~8:1, dissolving them in a low-boiling organic solvent, and then adding an siRNA MMP9-containing DEPC aqueous solution according to a molar ratio of the cationic lipid to the amino group / phosphate group in the siRNA of 1:1~8:1, ultrasonicating and vacuum concentrating to obtain a nanocomposite containing the antioxidant and the siRNA.

2. The method for preparing a nano-complex containing an antioxidant and siRNA according to claim 1, wherein, In step 1), the amino-containing antioxidant is one of 4-amin-2,2,6,6-tetramethylpiperidinooxy, spermine and superoxide dismutase; and / or, the compound containing a phenylboronic acid structure is one or a combination of two or more of 2-carboxyphenylboronic acid, 3-carboxyphenylboronic acid and 4-carboxyphenylboronic acid; and / or, the onium salt amide coupling agent is one of TBTU, HATU and HBTU.

3. The method for preparing the nanocomposite containing 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 the nanocomposite containing 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 organic solvent is one or a combination of two or more of chloroform, dichloromethane, ethyl acetate and methanol.

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

1.

6. The method of claim 1-5, wherein the preparation of the nanocomplex containing antioxidant and siRNA is characterized by, Step 1) specifically comprises: dissolving the amino-containing antioxidant and the compound containing a phenylboronic acid structure in N,N-dimethylformamide, adding the onium salt amide coupling agent after complete dissolution, and purifying the antioxidant with a phenylboronic acid bond through column chromatography after 24~48 hours of reaction at 40~45 °C.

7. The method of claim 1-5, wherein the preparation of the nanocomplex containing antioxidant and siRNA is characterized by, Step 2) specifically comprises: dissolving the antioxidant with a phenylboronic acid bond and the α-aminophosphonate derivative lipid containing catechol in methanol according to a molar ratio of 1:1, and obtaining the antioxidant-coupled lipid after 6~12 hours of reaction at 40~45 °C.

8. The method of claim 1-5, wherein the preparation of the nanocomplex containing antioxidant and siRNA is characterized by, Step 3) specifically comprises: dissolving the antioxidant-coupled α-aminophosphonate derivative lipid and the commercial cationic lipid in chloroform, mixing them according to a mass ratio of 1:1, adding an siRNA MMP9-containing DEPC aqueous solution according to a molar ratio of the amino group / phosphate group in the commercial cationic lipid to the siRNA of 3:1, and ultrasonicating and vacuum concentrating to obtain the nanocomposite containing the antioxidant and the siRNA.

9. A nanocomposite, characterized by, The method is prepared by any one of claims 1-8. The method is prepared by any one of claims 1-8.

10. Use of the nanocomposite of claim 9 in the preparation of a medicament for the repair of skin wounds in diabetes.

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