MMPs inhibitor and application thereof

Abstract

The invention relates to an MMPs inhibitor and application thereof, and belongs to the technical field of medicines.The MMPs inhibitor is prepared by the steps that siRNA capable of efficiently silencing MMPs is obtained through design and verification, then tea polyphenol nanoparticles with strong anti-inflammatory and anti-oxidation effects are adopted to carry two kinds of siRNAs capable of silencing matrix metalloproteinases MMP-2 and MMP-9 respectively, and the siRNAs with the strong anti-inflammatory and anti-oxidation effects are obtained; a novel multifunctional nano preparation TPNs-siRNA is constructed by using the siRNA as a template. Tests prove that the TPNs and the siRNAs can synergistically exert an efficient treatment effect, can be passively targeted to a lesion injury part, can slow down the progress of the disease course by scavenging free radicals and inflammatory factors, inhibiting vascular calcification and apoptosis and other mechanism levels, and can achieve the purpose of treating diseases such as abdominal aortic aneurysm, atherosclerosis, skin photoaging and the like in multiple dimensions; especially, the traditional Chinese medicine composition has a relatively good effect on treating AAA.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a MMPs inhibitor and application thereof. Background Art

[0002] Abdominal aortic aneurysm (AAA) is a degenerative aortic disease with a high mortality rate. It is more common in elderly male patients. The main manifestation is the local expansion of the abdominal aorta to form a tumor. It is more common in the infrarenal abdominal aorta segment. There are often no obvious symptoms before the tumor ruptures, but once it ruptures, the mortality rate is extremely high. At present, surgery is still the main means of treating this complex multifactorial disease, but due to the limitation of surgical indications (>5cm), there are also certain limitations and shortcomings. For patients with smaller AAA (3-5cm), the management focus is to prevent rupture and adopt a regular monitoring strategy. This "vigilant monitoring" strategy for small AAA patients not only causes great mental stress and economic burden to patients, but also has a certain risk of rupture due to the uncontrollable expansion rate of the tumor. However, there is currently no drug that effectively controls and slows down the expansion of AAA for clinical use, so there is an urgent need to develop safe and effective drugs to treat AAA.

[0003] Studies have shown that matrix metalloproteinases (MMPs) are important regulatory factors affecting the pathogenesis of AAA. The imbalance between matrix metalloproteinases and tissue inhibitors of metalloproteinases in the body causes their overproduction, degradation and destruction of ECM. Under inflammatory conditions, MMPs can cause the destruction of collagen fibers and elastic fibers in the media and adventitia of the abdominal aorta, weaken the elasticity of the arterial wall, and destroy the integrity of the aortic wall. Various types of MMPs are expressed in AAA tissues, among which MMP-2 and MMP-9 are the most widely studied and have always been potential targets for interventional treatment strategies for AAA. Systemic treatment with related MMPs inhibitors has shown its unique therapeutic effect in animal models, but due to poor efficacy when used at low doses and many systemic side effects when used at high doses, many studies have not been successfully transformed into clinical applications.

[0004] The role of small interfering RNA (siRNA) technology in the treatment of different diseases has been widely recognized. The use of siRNA to specifically silence target genes and interfere with the expression of MMP-2 and MMP-9 genes provides new opportunities for the treatment of AAA. Compared with MMPs inhibitors acting on the surface of target cells or the entire blood circulation system, siRNA can target intracellular mRNA levels and downregulate the protein expression of MMP-2 and MMP-9, which is more targeted. However, siRNA also has certain shortcomings, that is, it is easily degraded by nucleases in the plasma in the body before reaching the target cell, and it is highly hydrophilic, has a large molecular weight, and is negatively charged. It is difficult for free siRNA to cross the cell membrane barrier to enter the cell to exert its effect. Therefore, in the treatment of AAA, the delivery of siRNA often needs to rely on various carriers to achieve targeted administration to the AAA tumor site. TPNs are nanoparticles formed by self-aggregation of the tea polyphenol compound epigallocatechin gallate (EGCG). They have good biocompatibility, negligible low toxicity and immunogenicity, and have the properties of anti-inflammatory, free radical scavenging, and relieving oxidative stress. They are used to study the treatment of diseases. Summary of the invention

[0005] In view of the above problems, the purpose of the present invention is to provide an MMPs inhibitor and its application. The present invention obtains siRNA that can effectively silence MMPs by designing and verifying, and then prepares tea polyphenol self-polymerized nanoparticles TPNs-siRNA carrying siRNA according to the pathological change characteristics of AAA. The nanoparticles can combine the biological activities of TPNs in anti-oxidation, anti-inflammatory, and promotion of macrophage repolarization with the specific silencing of MMP-2 and MMP-9 molecules by siRNAs, and synergistically exert their efficient therapeutic effects. At the same time, it can passively target the lesion and damage site of AAA, slow down the progression of AAA by scavenging free radicals and inflammatory factors, inhibiting vascular calcification and apoptosis, and achieve the purpose of treating AAA in multiple dimensions.

[0006] In order to achieve the above object, the specific scheme adopted by the present invention is:

[0007] In a first aspect, an MMPs inhibitor includes an siRNA that silences MMP-2 and an siRNA that silences MMP-9, wherein the siRNA that silences MMP-2 is any one of siMMP-2-1, siMMP-2-2, and siMMP-2-3, and the siRNA that silences MMP-9 is any one of siMMP-9-1, siMMP-9-2, and siMMP-9-3; The nucleic acid sequence of the siMMP-2-1 is shown in SEQ ID NO: 1 and SEQ ID NO: 2; The nucleic acid sequence of the siMMP-2-2 is shown in SEQ ID NO: 3 and SEQ ID NO: 4; The nucleic acid sequence of the siMMP-2-3 is shown in SEQ ID NO: 5 and SEQ ID NO: 6; The nucleic acid sequence of the siMMP-9-1 is shown in SEQ ID NO: 7 and SEQ ID NO: 8; The nucleic acid sequence of the siMMP-9-2 is shown in SEQ ID NO: 9 and SEQ ID NO: 10; The nucleic acid sequence of the siMMP-9-3 is shown in SEQ ID NO:11 and SEQ ID NO:12.

[0008] As a further optimization of the above technical solution, the MMPs inhibitors include siMMP-2-1 and siMMP-9-3.

[0009] In the second aspect, the use of the above-mentioned MMPs inhibitor in the preparation of a nanoformulation for targeted delivery of therapeutic drugs. Further, the therapeutic drug includes but is not limited to drugs or preparations for treating abdominal aortic aneurysm, atherosclerosis or anti-skin photoaging. Further, the nanoformulation is to carry the matrix metalloproteinase MMPs inhibitor on a delivery carrier, and the delivery carrier is a TPNs carrier. Further, the nanoformulation is prepared by the following preparation method: in a HEPES buffer with a pH of 8.0, after adding the EGCG solution, the same volume of siMMP-2-1 and siMMP-9-3 solutions are added and pre-stirred for 30 minutes, and then the MnCl2 solution is slowly added dropwise, and vigorously stirred for 2.5 hours at a constant temperature of 37°C, the reaction solution is taken, centrifuged, the supernatant is discarded, and the TPNs-siRNA nanoformulation is prepared after washing with HEPES buffer.

[0010] Thirdly, the application of MMPs inhibitors and TPNs in synergistically inhibiting cellular oxidative stress.

[0011] In the fourth aspect, the application of the synergistic effect of MMPs inhibitors and TPNs in improving the targeting of in vivo delivery of therapeutic drugs. Further, the therapeutic drugs include but are not limited to drugs or preparations for treating abdominal aortic aneurysm, atherosclerosis or anti-skin photoaging, and the abdominal aortic aneurysm therapeutic drug is a nanoformulation of MMPs inhibitors carried on a TPNs carrier.

[0012] Beneficial effects: The present invention designs and obtains siRNAs for silencing MMPs for the two targets of MMP-2 and MMP-9, and verifies the efficiency of siRNAs in silencing MMP-2 and MMP-9 by siRNA cell transfection and RT-qPCR technology. On this basis, TPNs-siRNA nanoformulations are synthesized, which have both the efficacy of TPNs carrier and siRNA to synergistically exert anti-AAA effects. At the cellular level, TPNs-siRNA can be taken up by cells, remove the excessive RONS produced in LPS-induced macrophages and H2O2-induced vascular smooth muscle cells, inhibit macrophages from secreting inflammatory factors, promote the differentiation of macrophage M1-M2 phenotypes, reduce the expression of matrix metalloproteinases MMP-2 and MMP-9, and inhibit calcification and apoptosis of vascular smooth muscle cells. In the mouse AAA model, TPNs-siRNA can target AAA lesions, inhibit vascular calcification and tissue apoptosis, reduce the expression of inflammatory factors, MMP-2 and MMP-9, and reduce the level of ROS in local tissues, with good therapeutic effects. In vitro safety evaluation shows that TPNs-siRNA has good biosafety. The above experiments confirm the role of TPNs-siRNA in the treatment of AAA, which has very important practical significance and broad application prospects in the field of medicine. In addition, due to the in vitro and in vivo biological effects exhibited by the TPNs-siRNA of the present invention, it can be used not only to treat AAA, but also to treat other diseases or conditions, such as atherosclerosis, and also has the effect of delaying skin aging and treating photoaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Figure 2 is a graph showing the silencing efficiency of related siRNA on (A) MMP-2 and (B) MMP-9.

[0014] Figure 2 The figure is a result of gel electrophoresis experiment to investigate the encapsulation capacity of TPNs for siRNA, where (A) is the gel electrophoresis of FAM fluorescently labeled siRNA. The left side is the control channel of FAM-siRNA solution gel electrophoresis, and the right side is the supernatant gel electrophoresis channel after centrifugation of TPNs-siRNA solution. (B) is the quantitative result of (A) using Image J software.

[0015] Figure 3 These are the results of DLS investigation of TPNs-siRNA (A) particle size distribution and (B) ξ potential.

[0016] Figure 4 This is a graph showing the protection results of TPNs on siRNA.

[0017] Figure 5The following are the results of the scavenging activity of TPNs-siRNA at a series of concentrations on DPPH·. (A) UV spectra of TPNs-siRNA at a series of concentrations incubated with DPPH solution. (B) Kinetic curves of scavenging DPPH· by TPNs-siRNA at a series of concentrations. (C) Scavenging rates of DPPH by TPNs-siRNA at different concentrations.

[0018] Figure 6 The following are the results of the scavenging activity of TPNs-siRNA at a series of concentrations on ABTS+. (A) UV spectra of TPNs-siRNA at a series of concentrations incubated with ABTS+· solution. (B) Kinetic curves of ABTS+· scavenging by TPNs-siRNA at a series of concentrations. (C) The scavenging rate of ABTS+· by TPNs-siRNA at different concentrations.

[0019] Figure 7 The following are the results of the activity of TPNs-siRNA in scavenging ·OH: (A) UV absorption spectra of TPNs-siRNA incubated with ·OH reagent at a series of concentrations; (B) ·OH scavenging rates of TPNs-siRNA at a series of concentrations.

[0020] Figure 8 The following are the results of the ·O2- scavenging activity of TPNs-siRNA with a series of concentrations: (A) UV absorption spectra of the incubation of TPNs-siRNA with a series of concentrations and ·O2- reagent; (B) ·O2- scavenging rates of TPNs-siRNA with a series of concentrations.

[0021] Fig. 9 The following are the results of the activity of TPNs-siRNA in removing NO: (A) UV absorption spectra of TPNs-siRNA incubated with NO reagent; (B) NO removal rate of TPNs-siRNA in series of concentrations.

[0022] Fig.10 The results of RAW264.7 uptake of TPNs-siRNA are shown in Figure 2. (A) Laser confocal images of RAW264.7 and RAW264.7 uptake of TPNs-siRNA after LPS activation. Scale bar = 10 μm. (B) Flow cytometry results. (C) Mean fluorescence intensity quantitative graph.

[0023] Fig.11 The results of VSMCs uptake of TPNs-siRNA are shown in Figure 2. (A) Laser confocal images of VSMCs and VSMCs stimulated with H2O2 uptake of TPNs-siRNA. Scale bar = 10 μm. (B) Flow cytometry results. (C) Mean fluorescence intensity quantitative graph.

[0024] Fig.12The laser confocal images of co-localization of TPNs-siRNA and lysosomes, (A) TPNs-siRNA behavior trajectory in RAW264.7. (B) TPNs-siRNA behavior trajectory in VSMCs. Scale bar = 20 μm. (Blue: cell nucleus; Green: TPNs-siRNA; Red: lysosome).

[0025] Fig.13 It is the fluorescence image of TPNs-siRNA depleting RONS in RAW264.7 cells, scale bar = 100 μm.

[0026] Fig.14 The flow cytometry results and fluorescence quantification graphs of TPNs-siRNA clearing RONS in RAW264.7 cells, where: top: flow cytometry distribution histogram; bottom: corresponding mean fluorescence intensity graph.

[0027] Fig.15 It is the fluorescence image of TPNs-siRNA eliminating intracellular RONS in VSMCs, scale bar = 100 μm.

[0028] Fig.16 The flow cytometry results and fluorescence quantification graphs of TPNs-siRNA clearing intracellular RONS in VSMCs, where: top: flow cytometry distribution histogram; bottom: corresponding average fluorescence intensity graph.

[0029] Fig.17 Figure 2 shows the levels of inflammatory factors IL-1β (A), IL-6 (B) and TNF-α (C) in different treatment groups.

[0030] Fig.18 The results of the expression levels of RAW264.7 M1 markers and M2 markers in different treatment groups are shown in Figure 1. (A) Immunofluorescence images of iNOS and Arg-1 in RAW264.7 in different treatment groups. Scale bar = 100 μm. (B) RT-qPCR to investigate the expression levels of CD206, Arg-1, CD80 and iNOS genes.

[0031] Fig.19 The results of intracellular MMP-2 and MMP-9 in RAW264.7 cells in different treatment groups are shown in Figure 1. (A) RT-qRCP detects the expression level of MMP-2 gene. (B) RT-qRCP detects the expression level of MMP-9 gene. (C) ELASA detects the expression level of MMP-2 protein. (D) ELASA detects the expression level of MMP-9 protein.

[0032] Fig. 20The results of calcium deposition and cell viability detection of VSMCs in different treatment groups are shown in Figure 1. (A) Optical microscopy of calcium deposition in VSMCs. Scale bar = 100 μm. (B) Quantitative results of calcium deposition in VSMCs. (C) Cell viability of VSMCs after inducing calcium deposition.

[0033] Fig.21 The results of VSMCs apoptosis detection and cell viability detection in different treatment groups are shown in Figure 1, where (A) is the flow cytometry of VSMCs apoptosis. (B) is the quantitative result of VSMCs apoptosis. (C) is the cell viability of VSMCs after apoptosis induction.

[0034] Fig. 22 This is a general picture of the abdominal aorta of the two groups of mice.

[0035] Fig.23 The results of TPNs-Cy5.5-siRNA targeting in the abdominal aorta of mice are shown in Figure 1. (A) In vitro fluorescence imaging of TPNs-Cy5.5-siRNA in the abdominal aorta of mice. (B) Quantification of the mean fluorescence intensity of the abdominal aorta. (C) Distribution of TPNs-Cy5.5-siRNA in the main organs of mice. (D) Quantification of the mean fluorescence intensity in the liver and kidneys.

[0036] Fig.24 The following are the results of the retention time of TPNs-Cy5.5-siRNA in AAA mice. (A) In vitro fluorescence imaging of TPNs-Cy5.5-siRNA in the abdominal aorta of mice at different times, (B) quantification of the average fluorescence intensity of the abdominal aorta. (C) Distribution of TPNs-Cy5.5-siRNA in the main organs of mice at different times. (D) Quantification of the average fluorescence intensity in the liver and kidneys.

[0037] Fig.25 The results of tissue distribution of TPNs-Cy5.5-siRNA in the abdominal aorta of mice are shown in Figure 1. (A) Fluorescence microscopy images show the distribution of TPNs-Cy5.5-siRNA in the abdominal aorta tissue of the sham operation group. (B) Immunofluorescence analysis of co-localization of CD31+ endothelial cells, CD68+ macrophages, and α-SMA+ vascular smooth muscle cells with TPNs-Cy5.5-siRNA in aneurysms. Scale bar = 50 μm.

[0038] Fig.26 These are ultrasound images of the transverse section (upper) and longitudinal section (lower) of the abdominal aorta after treatment with different preparations.

[0039] Fig. 27 This is the result of color Doppler ultrasound measurement of the maximum diameter of the mouse abdominal aorta after treatment with different preparations.

[0040] Fig.28Comparison of (A) calcification and (B) H2O2 content in abdominal aorta tissue after treatment with different preparations.

[0041] Fig.29 This is the fluorescence imaging of ROS in abdominal aorta tissue after treatment with different preparations.

[0042] Fig.30 This is a comparison of the (A) MMP-2 and (B) MMP-9 levels in abdominal aorta tissue after treatment with different preparations.

[0043] Fig.31 This is a comparison chart of the contents of (A) IL-1β, (B) IL-6 and (C) TNF-α in abdominal aorta tissue after treatment with different preparations.

[0044] Fig.32 These are the results of H&E, EVG and Alizarin red staining of abdominal aorta sections after treatment with different preparations.

[0045] Fig.33 This is the TUNEL staining result of abdominal aorta sections after treatment with different preparations.

[0046] Fig.34 This is a graph showing the results of in vitro hemolysis experiments of two nanoformulations. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0048] Example 1

[0049] 1. Materials and Methods

[0050] Cell lines: The cell lines used in this study were mouse macrophages (RAW 264.7) and mouse vascular smooth muscle cells (VSMCs), both purchased from Xiangya Medical Experiment Center, Central South University.

[0051] Experimental animals: Healthy 8-week-old C57BL / 6 male mice were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. and fostered in the Animal Experiment Center of Central South University (SPF grade). All animals were kept in accordance with animal ethics and the management regulations of the Central South University Experimental Animal Welfare Ethics Committee.

[0052] Unless otherwise specified, all reagents and materials used are commercially available, and all methods used are conventional operating methods.

[0053] Among them, the cell culture operations involved, including cell recovery, cell culture and passaging, cell counting, cell freezing and other operations are all routine operating methods.

[0054] Primers: Primers were designed using Primer 5 software and referenced to the NCBI online primer design tool (primer-blast, http: / / www.ncbi.nlm.nih.gov / tools / primer-blast) to search for target gene sequences.

[0055] Table 1 Specific sequence list of primers used.

[0056] 2. Design and Synthesis of siRNA

[0057] siRNA sequences (three in each group) were designed for the two targets of MMP-2 and MMP-9, respectively, and synthesized by Shanghai Genema Gene Co., Ltd. The specific nucleic acid information of the siRNA sequences is shown in Table 2 below.

[0058] Table 2 Basic information of nucleic acid

[0059] Note: Since in ST.26, t represents uracil in RNA sequences and thymine in DNA sequences, in the xml sequence table, U and T in the sequences in Table 2 above are both displayed as t.

[0060] 3. Verification of siRNA silencing effect experiment

[0061] 1. siRNA transfection

[0062] RAW264.7 cells were plated in 6-well plates (so that the cell confluence reached 60%-80% within 24 hours during transfection). After the cells adhered to the wall, 200 μl OPTI-MEM medium (Invitrogen Gibco, USA) was added to a 1.5 ml sterile centrifuge tube, and 10 μl GP-transfect-Mate transfection reagent (Shanghai Jima Gene Co., Ltd.) was added. Mix gently with a pipette and let stand at room temperature for 5 minutes. At the same time, 200 μl OPTI-MEM medium was added to another 1.5 ml sterile centrifuge tube, and 10 μl siRNA was added. Mix gently with a pipette and let stand at room temperature for 5 minutes. The transfection reagent-medium mixture was added dropwise to the siRNA-medium mixture and let stand at room temperature for 18 minutes. While the plate is still, change the medium in the 6-well plate with 1.6 ml of preheated fresh culture medium in each well, then add the transfection system to each well and culture the cells at 37°C. After transfection for 4-6 hours, discard the supernatant of the 6-well plate and replace with complete culture medium and culture for 48 hours.

[0063] 2Real-time PCR

[0064] (1) RNA extraction

[0065] After washing the cells in the 6-well plate 3 times with PBS, add 1 ml TRIzol UP to each well of the 6-well plate, place horizontally for 5 minutes, blow the cells with a pipette, transfer the mixed solution in each well to an enzyme-free EP tube, and add 200 μl RNAExtraction Agent to each EP tube. Use a pipette to repeatedly blow and aspirate until there is no obvious precipitation, shake at room temperature for 5 minutes, centrifuge at 4°C precooling, 12000 rpm, centrifuge for 15 minutes, draw 200 μl of the top RNA-containing aqueous supernatant, add an equal volume of isopropanol, vortex and let stand for 10 minutes, centrifuge at 4°C precooling, 12000 rpm, centrifuge for 10 minutes. Discard the supernatant, add 1 ml 75% ethanol (prepared with enzyme-free water and anhydrous ethanol, precooled at 4°C), centrifuge at 12000 rpm for 5 minutes, repeat 2 times, completely discard the supernatant and dry at room temperature. After being fully dried, it was dissolved in 10 μl of enzyme-free water, and the total RNA concentration was determined using an ultra-micro spectrophotometer and diluted to 200 ng / μL.

[0066] (2) RNA reverse transcription was performed using a reverse transcription kit purchased from Thermo Fisher Scientific, Inc., USA. The specific operation was performed according to the method provided in the kit manual to synthesize cDNA.

[0067] After mixing the RNA sample and Primer, incubate at 65°C for 5 minutes and quickly cool in a 4°C refrigerator for 30 seconds. Then mix with the remaining reagents and reverse transcribe in a PCR instrument according to the sequence of "25°C for 15 minutes; 42°C for 60 minutes; 70°C for 5 minutes; 4°C for 10 minutes". After the program is completed, use an ultra-micro spectrophotometer to measure the cDNA concentration and dilute to 500ng / μL.

[0068] (3) Real-time fluorescence quantitative PCR: A real-time fluorescence quantitative PCR kit is used to analyze the transcription of the target RNA.

[0069] After the sample reagents are mixed, gently mix them. Detect them in a real-time fluorescence quantitative PCR instrument according to the sequence of "95℃ 10 minutes 40 cycles; 95℃ 15 seconds; 60℃ 30 seconds; 72℃ 40 seconds". Calculate the relative expression of mRNA in each group according to the formula, RQ = 2 -ΔΔct .

[0070] Real-time PCR experiments were used to verify the target silencing efficiency, such as Figure 1It was shown that different siRNAs had different silencing effects on the target, but the specific siRNAs selected in this application had a silencing efficiency of more than 70%. Among them, the first sequence of siMMP-2 (5′-CAUACAGGAUCAUUGGUUATT-3′, 5′-UAACCAAUGAUCCUGUAUGTT-3′) had the most obvious silencing effect, and the third sequence of siMMP-9 (5′-GCACUGGGCUUAGAUCAUUTT-3′, 5′-AAUGAUCUAAGCCCAGUGCTT-3′) had the most obvious silencing effect, so these two sequences were used in subsequent experiments.

[0071] Example 2

[0072] 1. Preparation of TPNs / TPNs-siRNA

[0073] Weigh appropriate amount of EGCG or MnCl2·4H2O powder using a precision balance, fully dissolve them in 30% ethanol or enzyme-free water as solvent, and prepare 100 mmol / L stock solutions at room temperature for later use.

[0074] Preparation of 1TPNs

[0075] In the HEPES buffer at pH 8.0, EGCG solution was added and pre-stirred for 5 minutes, and then MnCl2 solution was slowly added dropwise, and vigorously stirred for 1 hour at a constant temperature of 37°C (the final concentrations of each component were HEPES 10mmol / L; EGCG 2.5mmol / L; MnCl2 2mmol / L). The final reaction solution was centrifuged at 16000 rpm for 10 minutes, the supernatant was discarded, and the solution was washed twice with HEPES buffer (10mmol / L, pH 7.4), and the precipitate was resuspended using an ultrasonic instrument and stored at 4°C for later use.

[0076] Preparation of 2TPNs-siRNA

[0077] After adding EGCG solution to HEPES buffer at pH 8.0, the same volume of siMMP-2 and siMMP-9 solutions were added and pre-stirred for 30 minutes, and then MnCl2 solution was slowly added dropwise and vigorously stirred for 2.5 hours at a constant temperature of 37°C. The rest of the preparation process remained unchanged.

[0078] Study on Physical Characterization of TPNs-siRNA

[0079] 1. Investigation of siRNA loading capacity

[0080] (1) The 5' end of the siRNA sequence was labeled with FAM green fluorescence. The preparation process of TPNs-Cy5.5-siRNA referred to the preparation of TPNs-siRNA.

[0081] (2) When preparing TPNs-FAM-siRNA, the amount of siRNA input was increased so that the final concentration of siRNA input in the reaction system was 1 μmol / L, 2 μmol / L, and 4 μmol / L, respectively. After the preparation, the supernatant was centrifuged and detected by polyacrylamide gel electrophoresis. After the electrophoresis, the gel was placed in a UV imager to obtain a fluorescent image, and the image was analyzed and quantified by Image J software to calculate the siRNA loading amount of the carrier. siRNA loading amount = siRNA input amount - supernatant siRNA amount.

[0082] The results are as follows Figure 2 As shown, different concentrations (1, 2, 4 μmol / L) of FAM-siRNA were added to synthesize TPNs-siRNA, and no obvious nucleic acid was found in the supernatant. Further grayscale value analysis by Image J software found that when the nucleic acid input concentration was 1, 2, and 4 μmol / L, the encapsulation rate was >80%. It can be seen that TPNs is an efficient nucleic acid delivery carrier with a strong ability to load siRNA. In subsequent studies, the siRNA dosage concentration of 2 μmol / L was selected for the preparation of TPNs-siRNA. After freeze-drying, the TPNs-siRNA concentration was calculated to be about 500 μg / mL.

[0083] 2 Particle size determination

[0084] Take 100 μl of NPs dispersed in HEPES buffer and use Marlven Nano ZS instrument to measure the particle size by dynamic laser scattering method ( Figure 3 ), and each sample was run in parallel for 3 times. The results showed that the particle size of TPNs-siRNA after entrapped siRNA was slightly larger (171.9±2.3nm) compared with TPNs (158.2±1.6nm).

[0085] 3 Potential measurement

[0086] Sample preparation method: same particle size, 100 μl NPs were transferred to the potential cell, and the zeta potential was measured using a Marlven Nano ZS instrument ( Figure 3 ), and each sample was operated in parallel for 3 times. The results showed that the zeta potential of TPNs (-32.2±0.6mV) and the zeta potential of TPNs-siRNA (-32.7±0.7mV) did not change significantly.

[0087] 4TPNs protection of siRNA

[0088] Different concentrations of FBS (10%, 20%, 40%) were mixed with equal volumes of free siRNA labeled with FAM fluorescence and TPNs-FAM-siRNA loaded with equal amounts of siRNA, and 13 μl of each was placed in a 37°C water bath for incubation for 3 hours, and then heated in a 95°C water bath for 10 minutes. 1.5 μl of 1 mol / LEDTA, 0.5 μL of Tris-HCl (300 mmol / L, PH 7.4) and 10 μl of 8 mol / L urea were added, vortexed and mixed, and set aside. The degradation of siRNA was observed by polyacrylamide gel electrophoresis. Figure 4 The results showed that the degradation of siRNA in the control group became more and more serious with the increase of FBS concentration. When the FBS concentration was 20%, almost all free siRNA was degraded, and there was no obvious degradation of siRNA in the TPNs-siRNA group. From this result, it can be seen that TPNs has a good protective effect on siRNA.

[0089] 5. Stability study

[0090] TPNs-siRNA was dispersed in different solvents (PBS, medium containing 10% FBS1640, saline, H2O and HEPES solution), and the DLS particle size of NPs was measured at different time points (0 hours, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours). The results showed that TPNs-siRNA can be stably present in PBS, medium containing 10% FBS1640, saline, H2O and HEPES solution, and the particle size did not change significantly.

[0091] 3. Study on the functional characterization of TPNs-siRNA

[0092] The experimental method of Mensor et al. using DPPH to detect antioxidant activity and the experimental method of Miller et al. using ABTS to detect antioxidant activity were used. + The experimental method for evaluating antioxidant activity was used to explore the total antioxidant activity of TPNs-siRNA at different concentrations. Different kits were used to detect the scavenging effects of TPNs-siRNA at different concentrations on hydroxyl radicals (·OH) and superoxide anion radicals (·O2 - ) and nitric oxide (·NO).

[0093] 1TPNs-siRNA has the ability to scavenge free radicals

[0094] (1) This study determined the scavenging ability of a series of concentrations of TPNs-siRNA on DPPH· (DPPH method). Figure 5The UV spectra of TPNs-siRNA (0-100 μg / mL) co-incubated with DPPH solution showed that its scavenging effect on DPPH was dose- and time-dependent. The experimental phenomenon pictures showed that with the increase of TPNs-siRNA concentration, the DPPH solution gradually faded. Figure 5 BC showed that when the concentration of TPNs-siRNA increased from 5 μg / mL to 100 μg / mL, its DPPH·scavenging rate increased from 52.32% to 85.31%.

[0095] (2) This study investigated the effects of a series of concentrations (0, 5, 10, 25, 50, 100 μg / mL) of TPNs-siRNA on ABTS + The quenching ability of Figure 6 As shown in A, different concentrations of TPNs-siRNA had an effect on ABTS + The scavenging ability is dose- and time-dependent. When the concentration of TPNs-siRNA increases from 5μg / mL to 100μg / mL, its DPPH scavenging rate increases from 52.32% to 85.31%. When the dosage of TPNs-siRNA is 25μg / mL, the scavenging efficiency can reach 68.49% within 5 minutes, and most of ABTS can be basically cleared within 20 minutes. +· ( Figure 6 BC), showing rapid and effective antioxidant activity.

[0096] 2TPNs-siRNA has ·OH scavenging activity

[0097] The hydroxyl radical assay kit (Nanjing Jiancheng Biological Company) was used to detect the scavenging activity of TPNs-siRNA on ·OH. The basic principle is to generate ·OH through the Fenton reaction, which forms a red substance after incubation with the color developer, and its color is proportional to the amount of ·OH. The specific operation steps are as follows: dilute the TPNs-siRNA concentration to 0, 5, 10, 25, 50, 100, and 200 μg / mL respectively and detect according to the instructions of the kit. The experimental results are shown in the figure. Figure 7 As shown in the figure, when 10 μg / mL of TPNs-siRNA was used, a 63.17% clearance effect could be achieved, and when the concentration was increased to 100 μg / mL, almost all ·OH could be cleared.

[0098] 3TPNs-siRNA has the effect of clearing O2 - Activity

[0099] Use suppression and generation of O2 - The assay kit (Nanjing Jiancheng Biological Company) was used to measure the effect of different concentrations of TPNs-siRNA (5-200 μg / mL) on·O2 -The kit simulates xanthine oxygenase and xanthine to generate O2 - , and then incubated with a color developer to form a purple substance, the color of which is similar to that of O2 - is proportional to the amount. Figure 8 As shown in Figure 2, when the concentration of TPNs-siRNA was 100 μg / mL, it had an effect on the oxygen - The clearance rate can reach 63.57%, which is obviously dose-dependent.

[0100] 4TPNs-siRNA has NO scavenging activity

[0101] The NO scavenging activity of TPNs-siRNA on NO was determined using a NO assay kit (Shanghai Bio-Tech Co., Ltd.). Fig. 9 As shown, it was found that the use of 25 μg / mL TPNs-siRNA could achieve a 56.34% clearance effect, indicating its ability to clear NO.

[0102] Based on the above research results, TPNs loaded with siRNA does not affect the antioxidant effect of TPNs. It still has the activity of scavenging free radicals at the in vitro test tube level and shows the powerful antioxidant ability of TPNs-siRNA.

[0103] IV. Cytotoxicity Study

[0104] RAW264.7(1×10 4 / well) and VSMCs (5×10 3 / well) were plated on a 96-well plate and placed in an incubator for 12 hours. Use complete culture medium to dilute the sample to 0, 1, 2, 5, 10, 20, 50, 100, 200 μg / mL (calculated according to drug concentration). Discard the culture medium, set 6 replicate wells for each sample group, add 100 μl of diluted sample to each well, and incubate at 37°C for 24 hours. Discard the supernatant, wash twice with PBS, add 100 μl of 0.5 mg / mL MTT solution, incubate in an incubator for 4 hours, completely discard the MTT solution, add 150 μl DMSO to each well, shake on a shaker at 37°C for 10 minutes, and detect the absorbance of each well at 490 nm with an enzyme marker. Cell viability (%) = (OD value of the sample group - mean OD value of the zero-adjustment group) / (OD value of the blank group - mean OD value of the zero-adjustment group) × 100%.

[0105] The results showed that TPNs-siRNA had no toxicity to RAW264.7 and VSMCs at a concentration of 10-100 μg / mL, and the cell survival rate was almost unchanged, both above 90%. When the concentration was increased to 200 μg / mL, TPNs-siRNA still had no toxicity to RAW264.7, and the cell survival rate of VSMCs was only greater than 50%. This result provides a basis for the selection of TPNs-siRNA application doses in subsequent experiments. Combined with the results of previous studies, the TPNs-siRNA dosage was finally determined to be 25 μg / mL.

[0106] 5. Cellular uptake investigation

[0107] 1 Qualitative analysis of cellular uptake

[0108] (1) RAW264.7 was added at 1×10 5 Plant in confocal dishes, the volume of cell culture medium is 1ml / well. Set up blank group, LPS stimulation group, and no LPS stimulation group respectively. After the cells adhere to the wall and grow for 12 hours, the cell culture medium of the LPS stimulation group was replaced with complete culture medium containing 10μg / mL LPS for 48 hours. Then, the culture medium was discarded, and 25μg / mL TPNs-FAM-siRNA was added to the LPS stimulation group and the no LPS stimulation group for 4 hours. Discard the supernatant, wash 3 times with pre-cooled PBS, then add 4% paraformaldehyde to fix in the dark for 15 minutes, then discard the paraformaldehyde and add 1μg / mL DAPI application solution, incubate in the dark at room temperature for 10 minutes, wash 3 times with PBS and soak, and observe and take pictures under a confocal microscope (63x oil lens).

[0109] (2) VSMCs were seeded in confocal microplates at a density of 5×10 4 / well, the volume of cell culture medium was 0.5ml / well. A blank group, a H2O2 stimulation group, and a non-H2O2 stimulation group were set up respectively. After 12 hours, the cell culture medium of the H2O2 stimulation group was replaced with a complete culture medium containing 200μmol / L H2O2 for 6 hours. Then the culture medium was discarded, and 25μg / mL TPNs-FAM-siRNA was added and incubated for 4 hours. Wash three times with pre-cooled PBS, then add 4% paraformaldehyde and incubate at room temperature in the dark for 20 minutes to fix the cells, then discard the paraformaldehyde and add 1μg / mL LDAPI application solution, incubate at room temperature in the dark for 10 minutes, wash three times with PBS and soak in the confocal microscope for photography (63x oil objective).

[0110] 2 Quantitative analysis of cellular uptake

[0111] RAW264.7 and VSMCs in good condition were selected and 1×10 5 / well inoculated in a 6-well plate, the experimental grouping of the two cells was the same as "1 Qualitative analysis of cell uptake". After the drug treatment, the supernatant was discarded and washed twice with PBS. RAW264.7 cells were gently scraped off with a cell scraper to obtain a cell suspension. After VSMCs cells were completely digested with 200μl of pancreatic enzyme, an appropriate volume of culture medium was added to terminate the digestion, and the cell suspension was obtained after mixing. The two cell suspensions were collected separately, the centrifuge was set at 1000 rpm, the cell suspension was centrifuged for 5 minutes, PBS was washed again twice, and the dispersed cells were resuspended with 400μl PBS. The cell uptake of RAW264.7 and VSMCs was quantitatively analyzed by flow cytometry.

[0112] LPS was used to stimulate RAW264.7 to polarize to the M1 type to activate it, simulating the in vivo cell state of AAA. Here, we investigated the uptake of TPNs-siRNA by LPS-stimulated RAW264.7 and normal RAW264.7. In order to facilitate the observation of intracellular tracing imaging of nanoparticles, we labeled the 5′ end of the two siRNAs with green fluorescent FAM, synthesized FAM-labeled TPNs-FAM-siRNA preparations, and stained the RAW264.7 cell nucleus with DAPI solution to make it appear blue. Laser confocal imaging Fig.10 A shows that after TPNs-FAM-siRNA was incubated with normal RAW264.7 for 4 hours, obvious green fluorescence appeared in the FAM channel. The Merge diagram shows that the green fluorescence surrounds the outside of the cell nucleus marked by blue fluorescence, indicating that siRNA can enter RAW264.7 smoothly through the TPNs nanocarrier. Fig.10 BC flow cytometry results showed that the intracellular green fluorescence of activated RAW264.7 stimulated by LPS was significantly higher than that of normal RAW264.7, indicating that TPNs-siRNA can be taken up by M1 type RAW264.7 more effectively.

[0113] To simulate the in vivo cell state of AAA, we stimulated VSMCs with an appropriate dose of H2O2 and examined the uptake of TPNs-FAM-siRNA. Fig.11 A shows that after TPNs-FAM-siRNA was incubated with VSMCs for 4 hours, a large amount of green fluorescent substances appeared in the cells, indicating that siRNA can successfully enter VSMCs under the protection of TPNs. Fig.11 BC flow cytometry results showed that the uptake of nanoparticles by VSMCs stimulated by H2O2 was greater than that by normal VSMCs ( Fig.11 ), which shows that TPNs-siRNA has a high targeting effect on VSMCs in oxidative stress response.

[0114] 6. Lysosomal escape assay

[0115] RAW264.7 and VSMCs in good condition were selected and inoculated into confocal microplates respectively, and diluted to a density of 5×10 4 / ml cell suspension, inoculated into confocal dishes with 1ml per well. After culturing in the cell culture incubator for 12 hours, discard the culture medium. Add 1ml of TPNs-FAM-siRNA diluted with culture medium to each dish (the concentration is 25μg / mL based on drug concentration). One dish was incubated for 2 hours and then the culture was terminated. The culture medium was discarded and washed 3 times with PBS. 500μl of serum-free 1640 culture medium containing lysosomal red fluorescent probe was added. After incubation in the incubator for 50 minutes, the PBS was washed 3 times to complete lysosomal staining. Subsequently, the nuclear stain DAPI application solution was added, and the nuclear staining was completed after incubation and staining for 10 minutes and then washed 3 times with PBS. After completing the above operations, the photos were observed under a laser confocal microscope. The incubation time of TPNs-FAM-siRNA in the other two dishes was 4h and 8h, and the other operations were the same as above.

[0116] The results of cell uptake showed that TPNs-siRNA can be taken up by RAW264.7 and VSMCs, but the final effect of siRNA requires siRNA to escape from the lysosome to the cytoplasm. Lysosomal probes were used to co-localize TPNs-siRNA and lysosomes to investigate the lysosomal escape ability of TPNs-siRNA. Laser confocal microscopy was used to observe and photograph the co-localization of cell lysosomes (red fluorescence) and TPNs-siRNA (green fluorescence). The experiment found that whether it was RAW264.7 ( Fig.12 A) or VSMCs ( Fig.12 B), after 4 hours of incubation, TPNs-siRNA was mainly present in lysosomes (yellow area), and after 8 hours, green fluorescence gradually appeared at the edge of the nucleus, indicating that siRNA in TPNs-siRNA can successfully escape from lysosomes into the cell cytoplasm. At the same time, the results also suggest that RAW264.7 and VSMCs may engulf TPNs-siRNA through the lysosomal pathway.

[0117] 7. Free radical scavenging verification

[0118] 1 Establishment of a cell model with high RONS levels

[0119] (1) RAW264.7 cells were stimulated with 10 μg / mL LPS for 48 h to construct a macrophage inflammatory cell model with high RONS levels.

[0120] (2) VSMCs were stimulated with 200 μmol / L H2O2 for 6 h to construct a vascular smooth muscle cell model with high RONS levels.

[0121] Qualitative study on free radical scavenging by 2TPNs-siRNA

[0122] RAW264.7 and VSMCs cells were passaged and the cells were counted so that the number of cells per well reached 5×10 4 , inoculated in 24-well plates, and the culture medium was gently blown with a pipette to evenly distribute the cells, and then placed in an incubator for overnight incubation. The experiment was divided into blank control group, TPNs-siRNA group, model group, and treatment group.

[0123] (1) After RAW264.7 cells adhered to the wall, the supernatant was discarded. The model group was stimulated with medium containing 10 μg / mL LPS for 48 hours, and the treatment group was stimulated with medium containing 25 μg / mL TPNs-siRNA for 24 hours. The treatment group was stimulated with medium containing 10 μg / mL LPS for 48 hours and then supplemented with medium containing 25 μg / mL TPNs-siRNA. The blank control group did not require any treatment and only had the cell medium replaced.

[0124] (2) After VSMCs adhered to the wall, the supernatant was discarded. The model group was stimulated with medium containing 200 μmol / L H2O2 for 6 h, and the treatment group was stimulated with medium containing 25 μg / mL TPNs-siRNA for 24 h. After 6 h of stimulation with medium containing 200 μmol / L H2O2, the treatment group was supplemented with medium containing 20 μg / mL TPNs-siRNA. The blank control group did not require any treatment and only had the cell medium replaced.

[0125] After different treatments were performed on the well plates of the two cells, the culture medium was discarded, and the plates were washed with PBS three times. Three free radical probes ROS-ID, DCFH-DA, DHE, HPF, and DAF-FM DA (diluted with phenol red-free 1640 medium containing 10% FBS to concentrations of 5 μmol / L, 30 μmol / L, 5 μmol / L, 20 μmol / L, and 5 μmol / L, respectively) were added to the well plates of each cell, and the incubation time in the incubator was 30 minutes for ROS-ID, 30 minutes for DCFH-DA, 30 minutes for DHE, 45 minutes for HPF, and 30 minutes for DAF-FM DA, respectively. The supernatant was discarded, the plates were washed with PBS three times, 500 μl of phenol red-free 1640 medium was added, and the plates were observed and photographed under a fluorescence microscope.

[0126] Quantitative study on free radical scavenging by 3TPNs-siRNA

[0127] RAW264.7 and VSMCs were plated at 1×10 per well. 5The cells were seeded at a density of 100 μl in 6-well plates and incubated overnight before being treated in different experimental groups. The steps were the same as those for the qualitative study. After probe loading, the cells were washed three times with PBS. The RAW264.7 cells were gently scraped with a cell scraper to obtain a cell suspension. 200 μl of trypsin was added to each well of the VSMCs plate and digested for 1 min. 1 ml of phenol red-free culture medium was added to terminate the digestion to obtain a cell suspension. The cells were centrifuged at 1000 rpm for 5 min and washed twice with PBS. 400 μl of PBS was added to disperse the two types of cells and quantitatively analyzed by flow cytometry.

[0128] 4 Results: TPNs-siRNA has the activity of clearing intracellular RONS

[0129] 4.1TPNs-siRNA has the activity of clearing RONS in RAW264.7 cells

[0130] Five probes, ROS-ID, DCFH-DA, DHE, DAF-FM DA, and HPF, were used to investigate the effects of TPNs-siRNA on RONS (total ROS, H2O2, ·O2 - , NO, OH / ONOO - ). The results showed that the RAW264.7 model group treated with LPS showed stronger fluorescence than the blank control group and the control group treated with TPNs-siRNA alone, which means that LPS-treated RAW264.7 can produce a large amount of free radicals and the cells are in a state of severe oxidative stress. After TPNs-siRNA treatment, the fluorescence signal was significantly reduced ( Fig.13 ). Combined Fig.14 The flow cytometry and quantitative results of different treatment groups showed that after LPS stimulation, the levels of total ROS and NO in cells were significantly increased, and the levels of H2O2, ·O2 - and OH / ONOO - The levels also increased to varying degrees, and after TPNs-siRNA treatment, the contents of each RONS in the cells were significantly reduced.

[0131] 4.2TPNs-siRNA has the activity of clearing RONS in VSMCs

[0132] Similarly, five probe stainings were used to study the important RONS in cells through fluorescence microscopy. The VSMCs cells treated with H2O2 showed stronger fluorescence than normal cells, indicating that the cells were in a state of severe oxidative stress ( Fig.15 ). After TPNs-siRNA treatment, the fluorescence signal was significantly reduced due to its ability to effectively scavenge free radicals. Fig.16In the experiment, flow cytometry and quantitative detection showed that after H2O2 stimulation, the corresponding free radical level could be significantly increased, and TPNs-siRNA treatment could significantly eliminate the excessive free radicals in the cells. When detecting the intracellular H2O2 content, the H2O2 in the model group increased significantly, which may be related to the stimulation of H2O2 solution in our cell model. However, after TPNs-siRNA treatment, H2O2 was significantly reduced.

[0133] The above results show that TPNs loaded with siRNA can still stably exert their antioxidant capacity and have a good free radical scavenging effect.

[0134] 8. Anti-inflammatory Effects

[0135] Select RAW264.7 in good condition and seed them in 6-well plates at a density of 1×10 5 / well, the volume of complete cell culture medium was 2 ml. After the cells adhered to the wall, except for the blank control group, all other groups were stimulated with 10 μg / mL LPS for 48 hours. After the TPNs group and TPNs-siRNA group discarded the culture medium, 25 μg / mL TPNs and 25 μg / mL TPNs-siRNA were added and incubated for 24 hours. The cell supernatant was then collected and the content was determined according to the operating instructions of the ELISA detection kits for IL-1β, TNF-α and IL-6. The blank control cells in this experiment were not treated in any special way, and the cells in the positive control group were treated by adding only LPS stimulation. The other operating procedures were the same as those of the experimental group.

[0136] like Fig.17 As shown in the results, RAW264.7 cells pretreated with LPS can produce a large number of inflammatory cytokines. Compared with the model group, both TPNs-siRNA and TPNs can effectively downregulate IL-1β, IL-6 and TNF-α secreted by RAW264.7, indicating that they have certain anti-inflammatory activity. However, there are differences between TPNs-siRNA and TPNs in inhibiting inflammatory factors. TPNs-siRNA is more significant than TPNs, but the difference is not significant.

[0137] IX. Study on the effect of promoting macrophage polarization

[0138] 1 Immunofluorescence

[0139] RAW264.7 is set to density 2×10 5Cells were inoculated into 6-well plates with glass slides to make cell slides. The experiment was divided into four groups: blank control group, LPS group, LPS+TPNs group and LPS+TPNs-siRNA group. Cell treatment was as described in "VIII. Study on anti-inflammatory effect". After drug treatment, cells were washed twice with PBS, 500ul of PBS containing iNOS and Arg-1 antibodies was added to each well and incubated for 2 hours, washed twice with PBS, fixed with 4% paraformaldehyde for 10 minutes, and then DAPI nuclear dye application solution was added. After staining the nucleus for 5 minutes, PBS was washed again twice. After completion, fluorescence imaging was performed under a fluorescence microscope.

[0140] 2RT-qPCR technology

[0141] The experimental grouping and cell treatment methods were the same as above. After the drug incubation, the cells were collected to extract total RNA. Subsequent experiments referred to the "Real-time PCR" operation steps in Example 1, and the level of intracellular target mRNA was determined by PCR. CD206, Arg-1, CD80 and iNOS primers were purchased from Shanghai Baixin Company. The primer sequences are detailed in Table 1.

[0142] 3 Results: TPNs-siRNA promotes the repolarization of M1 macrophages to M2

[0143] The phenotypic changes of macrophages were studied by immunofluorescence staining of iNOS (M1 marker) and Arg-1 (M2 marker). Fig.18 As shown in A, after LPS stimulation, the red fluorescence signal of iNOS was significantly enhanced, while the green fluorescence signal of Arg-1 was very weak, indicating that RAW264.7 polarized to M1 type. After treatment with TPNs-siRNA and TPNs, the fluorescence of iNOS was significantly weakened, while the fluorescence of Arg-1 was enhanced, indicating that RAW264.7 repolarized from M1 type to M2 type. Fig.18 B, We further verified the expression of M1 markers (CD80 and iNOS) and M2 markers (CD206, Arg-1) at the mRNA level using RT-qRCP technology, and the results were consistent with Fig.14 The results showed that iNOS was inhibited and Arg-1 was increased.

[0144] 10. Study on the inhibitory effect of MMPs

[0145] 1ELISA test

[0146] Normal RAW264.7 cells were plated in 6-well plates at a density of 1×105 / well. The experimental grouping and cell treatment methods were the same as in “VIII. Study on anti-inflammatory effects”. After drug incubation, the cell supernatant was collected and centrifuged at 1000 rpm for 15 minutes. The contents of MMP-2 and MMP-9 were detected according to the instructions of the ELISA kit.

[0147] 2RT-qPCR technology

[0148] After different treatments, cells were collected to extract total RNA. Subsequent experiments were performed according to the operating procedures of "VIII. Study on anti-inflammatory effects". MMP-2 and MMP-9 primers were purchased from Shanghai Baixin Company. The primer sequences are shown in Table 1.

[0149] 3 Results: TPNs-siRNA has an anti-MMPs effect

[0150] The key to this study is that siRNA can continue to silence MMP-2 and MMP-9 under the protection of TPNs. To this end, we detected the levels of MMP-2 and MMP-9 secreted by RAW264.7 by RT-qPCR and ELISA. Fig.19 As shown in the figure, TPNs-siRNA continued to exert its anti-MMP-2 and MMP-9 effects at the cellular level, while TPNs may regulate and inhibit MMP-2 and MMP-9 through complex cellular mechanisms, but its effect was significantly weakened compared with TPNs-siRNA.

[0151] 11. Anti-VSMCs calcification study

[0152] Qualitative study on the inhibition of VSMCs calcification by 1TPNs-siRNA

[0153] Normal VSMCs were plated at 0.5 × 10 per well. 5 The cells were evenly inoculated in a 24-well plate at a density of 100 μg / mL. The culture medium was gently shaken to allow the cells to be evenly distributed. Then, the cells were placed in an incubator for 12 hours to allow the cells to fully adhere to the wall. After incubation, the supernatant was discarded, and 1 ml of complete culture medium containing 25 μg / mL TPNs 1640 and 25 μg / mL TPNs-siRNA1640 were added to the well plate and incubated with the cells for 24 hours. After replacement with 1 ml of fresh 1640 complete culture medium containing Ca / Pi (2 mmol / L CaCl2, 2.7 mmol / L NaH2PO4 and Na2HPO4 mixed solution, PH 7.4), the cells were incubated for another 24 hours. Wash with PBS three times, fix with 600 μl 4% paraformaldehyde for 10 minutes, stain with 0.2% (w / v) alizarin red solution for 30 minutes, and wash with PBS four times. Observe the calcification with an optical microscope and take pictures.

[0154] Quantitative study on the inhibition of VSMCs calcification by 2TPNs-siRNA

[0155] The experimental grouping and cell treatment methods were the same as above. After Ca / Pi stimulation for 24 hours, 1 ml of 10% cetylpyridinium chloride was added to each group of cells. The plate was shaken at a low speed for 10 minutes, and the absorbance at 405 nm was detected by an ELISA reader. The absorbance reflects the degree of calcification and can be used for quantitative statistical analysis of the degree of VSMCs calcification.

[0156] 3. Cell viability detection

[0157] After the cells were stimulated with Ca / Pi for 24 hours, the CCK-8 kit was used to detect the cell viability of VSMCs in each group.

[0158] 4 Results: Alizarin red can dye calcium-deposited cells into rose red. As shown in Figure 20A, compared with the cells in the normal group, the VSMCs in the model group had obvious calcium deposition. After treatment with TPNs and TPNs-siRNA, the redness of the cells in the model group weakened and the degree of calcification was reduced. Fig. 20 The quantitative results in B further illustrate the ability of TPNs and TPNs-siRNA to inhibit VSMCs calcification, supporting the light microscopy results. Fig. 20 C is the cell viability test of VSMCs in each experimental group. The results show that compared with the approximately 25% mortality rate of cells in the model group, the two nanoformulations can reduce the damage of VSMCs induced by Ca / Pi, and there is basically no cell death.

[0159] 12. Anti-VSMCs apoptosis study

[0160] 1VSMCs apoptosis detection

[0161] VSMCs growing in logarithmic numbers were cultured at 5 × 10 4 / cell density was evenly seeded on a 6-well cell culture plate and incubated overnight in a cell culture incubator to allow the cells to adhere. The experiment was divided into four groups: normal group, H2O2 group, H2O2+TPNs group and H2O2+TPNs-siRNA group. In the H2O2 group, only 600μmol / L H2O2 was added to the culture medium to incubate the cells for 6 hours. In the H2O2+TPNs group and H2O2+TPNs-siRNA group, 2ml of 25μg / mL TPNs and 2ml of 25μg / mL TPNs-siRNA solution were added to the cells respectively. After incubation for 24 hours, the cells were incubated with 600μmol / L H2O2 culture medium for 6 hours. According to the instructions of the apoptosis detection kit, the cells were washed twice with pre-cooled PBS, digested with EDTA-free trypsin to collect the cells, centrifuged at 2000 rpm for 5 minutes, discarded the supernatant, added 500ul of Binding buffer to resuspend the cells, and then added 5ul of Annexin V-APC and 5ul of Propidium Iodide in sequence, and mixed thoroughly. Incubate at room temperature in the dark for 15 minutes, observe and detect with a flow cytometer after 1 hour, and determine the fluorescence intensity using FlowJo V.10 software.

[0162] 2. Cell viability assay

[0163] 5×10 VSMCs were grown in logarithmic growth in each well. 3 The cells were evenly inoculated in a 96-well plate and incubated in a cell culture incubator for 12 hours to allow the cells to adhere to the wall. The experimental grouping and cell treatment methods were the same as above. After the corresponding time, PBS was washed and the drug-containing culture medium was removed. According to the instructions for the CCK8 detection kit, 100ul CCK8 solution was added to each well. The absorbance (OD) value at 450nm was analyzed by an enzyme-labeled instrument.

[0164] 3 Results: Studies have shown that ROS produced during oxidative stress can induce VSMCs apoptosis. To this end, we used high concentrations of H2O2 to stimulate VSMCs to verify whether TPNs and TPNs-siRNA have anti-apoptotic effects. The experimental results showed that the number of apoptotic and necrotic cells in the model group was much greater than that in the normal control group, indicating that high concentrations of H2O2 can cause VSMCs death ( Fig.21 B). After treatment with TPNs and TPNs-siRNA, the degree of cell apoptosis was alleviated to varying degrees ( Fig.21 A), suggesting that it may inhibit the apoptosis of VSMCs by scavenging intracellular oxidative free radicals.

[0165] 13. Establishment of AAA Animal Model

[0166] Healthy 8-week-old male C57BL / 6 mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.1 ml / 10 g). After successful anesthesia, the abdominal skin was prepared and the mice were fixed on a sterile operating table in a supine position. An incision was made along the midline of the abdomen to open the abdominal cavity. The abdominal aorta was separated under a stereomicroscope. The separation point was selected below the left renal vein, where the abdominal aorta and the inferior vena cava were loosely connected and easy to operate. The blood vessel diameter was measured with a vernier caliper. Sterile gauze was cut into small pieces of 0.5 cm × 0.5 cm. Soak in a CaCl2 solution prepared in 0.5 mol / L PBS; wrap the sterile gauze soaked in CaCl2 solution on the adventitia of the infrarenal abdominal aorta; remove the gauze after 15 minutes, use normal saline to thoroughly clean the abdominal cavity, and use sterile cotton balls to absorb the residual solution in the abdominal cavity; mice in the sham operation group followed the above surgical procedure, wrapped the infrarenal abdominal aorta with normal saline and incubated for 15 minutes; suture the wound with 4-0 needle sutures, place the animals on a warming pad, and feed them normally after waking up.

[0167] The AAA model was established by incubating the infrarenal abdominal aorta segment of C57BL / 6J mice with CaCl2 solution. After the experiment, the abdominal aorta was dissected and photographed under a stereo microscope. Fig. 22 This is a representative image of mouse AAA. Local aneurysmal dilatation of the abdominal aorta can be seen with the naked eye. The diameter of the blood vessels in the control group was 0.3 mm and that in the model group was 0.6 mm, indicating that the modeling was successful. This animal model simulates the prone lesion site of human AAA (the infrarenal abdominal aorta segment), and the tumor often presents a fusiform structure similar to human AAA.

[0168] 14. In vivo targeting studies

[0169] 1TPNs-Cy5.5-siRNA preparation

[0170] The 5' end of the siRNA sequence was labeled with Cy5.5 red fluorescence.

[0171] In vivo targeting of 2TPNs-Cy5.5-siRNA

[0172] Nine C57BL / 6 male mice were randomly divided into three groups (n=3): sham operation group (AAA - )+TPNs-Cy5.5-siRNA, model group (AAA + )+Cy5.5-siRNA and model group (AAA +)+TPNs-Cy5.5-siRNA. After the mouse AAA model was established, the mice were raised conventionally for 2 weeks and then injected with drugs through the tail vein. One group of AAA mice and sham-operated mice were injected with saline solution containing TPNs-Cy5.5-siRNA (50 μl / mouse), and the other group of AAA mice were injected with saline solution containing free Cy5.5-siRNA (50 μl / mouse). The injection dose of TPNs-Cy5.5-siRNA into the tail vein of mice was uniformly 25 mg / Kg, and the injection dose of Cy5.5-siRNA was 1 mg / Kg. Eight hours after administration, the mice in each group were euthanized, and the aortic tissue (thoracic aorta, abdominal aorta to the bifurcation of the iliac artery), heart, liver, spleen, lungs and kidneys were collected. After washing with sterile PBS, the organs of each group were placed in the IVISⅢ small animal in vivo imager for observation, and the fluorescence intensity was quantitatively analyzed.

[0173] from Fig.23 A results show that TPNs-Cy5.5-siRNA has strong targeting, and the fluorescently labeled nanomedicine mainly accumulates in the subrenal tumor-forming artery segment of mice ( Fig.23 B) The aortic arch showed partial fluorescence, which may be due to the residual fluorescent drug caused by insufficient saline irrigation of the aorta during the dissection of the free abdominal aorta of the mouse. No obvious fluorescence accumulation was observed in the aorta of AAA mice injected with free Cy5.5-siRNA and sham-operated mice injected with TPNs-Cy5.5-siRNA. After collecting and processing the abdominal aorta of the mouse, the heart, liver, spleen, lung, and kidney of the mouse were collected for IVIS photography. Fig.23 As shown in C, the nanomedicine is mainly concentrated in the liver, lungs and kidneys, and the fluorescence quantification of the liver and kidneys, the main metabolic organs of the drug, is performed ( Fig.23 D) No significant fluorescence aggregation was observed in the organs of mice injected with free siRNA solution, which may be due to its rapid metabolism or nucleic acid degradation. - (TPNs-Cy5.5-siRNA) mice and AAA + Compared with the (TPNs-Cy5.5-siRNA) mice, the fluorescence accumulation in the liver, lungs and kidneys was more concentrated, indicating that part of the TPNs-Cy5.5-siRNA had acted on the AAA site of the mice. - The fluorescence accumulation in mouse organs will be relatively weak.

[0174] Study on the time of 3TPNs-Cy5.5-siRNA acting on abdominal aorta

[0175] Fifteen C57BL / 6 male mice were randomly divided into five groups (n=3). Four groups of mouse AAA models were established according to step 3.4.18.1. The other group was used as a sham operation group. After 14 days, normal saline solution (50 μl / mouse) containing TPNs-Cy5.5-siRNA was injected through the tail vein. After 3h, 12h, 24h and 48h, the mice in each group were euthanized, and the rest of the treatment was the same as above.

[0176] Next, we investigated the retention effect and in vivo biodistribution of TPNs-siRNA in the abdominal aorta of AAA mice. Fig.24 A. Fig.24 As shown in B, as the administration time increases, the accumulation of TPNs-Cy5.5-siRNA in the abdominal aorta of mice gradually weakens. After 48 hours, almost no fluorescence can be seen gathering in the abdominal aorta. Fig.24 C. Fig.24 D shows that TPNs-Cy5.5-siRNA accumulates significantly in the liver, lungs, and kidneys, and as the administration time increases, the fluorescence gradually weakens. Twelve hours after administration, the fluorescence intensity of the liver and kidneys is significantly weakened, especially in the kidneys, suggesting that TPNs-siRNA may be metabolized to the body through the kidneys and liver.

[0177] Histological analysis of 4TPNs-Cy5.5-siRNA in AAA

[0178] Six C57BL / 6 male mice were divided into two groups (n=3): model group and sham operation group. 14 days after the establishment of AAA animal model, physiological saline solution containing TPNs-Cy5.5-siRNA (50μl / mouse) was injected through the tail vein. 8 hours later, the mice were euthanized, and the infrarenal abdominal aorta segments incubated with CaCl2 solution were collected, and the samples were embedded in OCT glue and sliced ​​into 10um. The slices were degummed to water, and the slices were first placed in distilled water for 5-10min, and blocked with 5% BSA for 60 minutes. The primary antibody of the target protein (CD31, CD68, α-SMA) was added dropwise, and incubated at 4℃ overnight. Washed with PBS 3 times, incubated with secondary antibody: 50-100ul anti-rabbit, rabbit, rabbit-IgG labeled fluorescent antibody was added dropwise, incubated at 37℃ for 90 minutes, and rinsed with PBS for 5 minutes 3 times. DAPI working solution was used to stain the nucleus for 10 minutes at 37℃, and rinsed with PBS for 5 minutes 3 times. Seal the slides with buffered glycerol and observe under a fluorescence microscope.

[0179] Blue fluorescence represents cell nuclei stained with DAPI, red fluorescence represents TPNs-Cy5.5-siRNA, and green fluorescence represents cells labeled with different antibodies. Fig.25A The results showed that no red fluorescence appeared in the Cy5.5 channel of the sham group, indicating that TPNs-siRNA did not accumulate in the tumor site of AAA. A large amount of red fluorescence was observed in the Cy5.5 channel of the model group ( Fig.25 B), TPNs-siRNA and α-SMA + Labeling of vascular smooth muscle cells and CD68 + The labeled macrophages showed different degrees of fluorescence co-localization, indicating that TPNs-siRNA could be effectively taken up by vascular smooth muscle cells and macrophages in the AAA tumor tissue, which strongly supported the above cell experiment results.

[0180] 15. In vivo anti-AAA study

[0181] 1. Dosing regimen for mice

[0182] Twenty-four male C57BL / 6 mice were randomly divided into 4 groups (n=6): normal group; AAA model group; AAA group + TPNs; AAA group + TPNs-siRNA. The mouse AAA model was established and routinely fed. Four weeks after surgical intervention modeling, the mice in the two treatment groups were given normal saline solution (50 μl / mouse) containing TPNs and TPNs-siRNA, respectively, at a dose of 25 mg / kg, through the tail vein. The normal group and the AAA model group were injected with a simple normal saline solution (50 μl / mouse) through the tail vein. The treatment lasted for 2 weeks, with injections twice a week.

[0183] 2 Ultrasound detection of mouse abdominal aorta

[0184] After the administration, the abdominal skin of the mice was prepared and hair removed. Under inhalation anesthesia, Vevo 2100 micro-ultrasound scanning was used to observe the morphology of the abdominal aorta and measure the maximum diameter of the infrarenal abdominal aorta.

[0185] like Fig.26 As shown, we observed the morphology of blood vessels from the coronal and sagittal planes of the abdominal aorta and found that compared with the normal group of mice, the infrarenal abdominal aorta segment of the model group mice was significantly enlarged and the vascular wall was calcified. After different drug treatments, the vascular diameter decreased to varying degrees, among which the vascular diameter reduction in the TPNs-siRNA treatment group was the most significant.

[0186] like Fig. 27 As shown, after the blood vessel diameter was measured and analyzed, it was found that the diameter of the abdominal aorta in the model group was about 1.5 times that of the normal group, while the diameter of the abdominal aorta in the TPNs and TPNs-siRNA treatment groups was reduced, and the reduction in blood vessel diameter in the TPNs-siRNA treatment group was the most obvious.

[0187] 3 Detection of calcification and H2O2 content in abdominal aorta tissue after treatment with different preparations

[0188] Vascular calcification and ROS produced by oxidative stress response are one of the main characteristics of abdominal aortic aneurysm. We used H2O2 in ROS as a representative, so we used corresponding kits to detect the calcification content and H2O2 content in abdominal aortic tissue homogenate, such as Fig.28 As shown in the figure, compared with the normal group, the calcium content and H2O2 level in the model group were significantly increased, and both nanoformulations could reduce the calcium deposition content and H2O2 level in AAA tissues. There was no difference between TPNs and TPNs-siRNA.

[0189] 4 Expression of ROS in abdominal aorta tissue after treatment with different preparations

[0190] We performed ROS staining on the blood vessels in the AAA tumor area and observed the changes of ROS in the diseased arteries ( Fig.29 ). The cell nucleus in the DAPI channel is blue, and the ROS in the Cy3 channel is red. The results showed that TPNs and TPNs-siRNA can reduce the accumulation of ROS in AAA tumors, and the therapeutic effect of the TPNs-siRNA group seems to be better.

[0191] 5. Determination of MMP-2 and MMP-9 content in abdominal aorta tissue after treatment with different preparations

[0192] To investigate whether siRNA exerts a strong silencing effect in the mouse AAA model as in the cell experiment, we used ELISA to detect the levels of MMP-2 and MMP-9 in AAA tissue homogenate. Fig.30 As shown, MMP-2 and MMP-9 in the model group were expressed at high levels. TPNs and TPNs-siRNA both showed the effect of reducing the two MMPs to a certain extent, but TPNs-siRNA carrying siRNA was more effective in reducing MMPs than TPNs.

[0193] 6. Contents of inflammatory factors in AAA tissue after treatment with different preparations

[0194] like Fig.31 As shown in the figure, ELISA kits were used to detect IL-1β, IL-6 and TNF-α in AAA tissue homogenate, and it was found that the levels of inflammatory factors were reduced after treatment with different nanoformulations. There was no significant difference in the anti-inflammatory effect of TPNs and TPNs-siRNA in AAA.

[0195] 7 Analysis of AAA tissue sections after treatment with different preparations

[0196] The morphological changes, calcification degree and elastic fiber degradation of AAA were observed by using H&E, EVG and Alizarin Red staining on AAA paraffin sections. Fig.32 As shown in the figure, compared with the model group, both TPNs and TPNs-siRNA can effectively improve AAA pathological changes. EVG staining results showed that the degradation of elastic fibers in both treatment groups was significantly alleviated. Alizarin red staining reflects the degree of tissue calcium deposition. The results showed that the model group had severe calcification, and both nanoformulations showed good anti-calcification effects.

[0197] Detection of apoptosis of abdominal aorta tissue cells after treatment with 8 different preparations

[0198] We performed TUNEL staining on abdominal aorta tissue sections, observed and photographed them under an optical microscope, and captured images with a computer at a magnification of 40×10. We also used Image-Pro Plus 6.0 image processing software to analyze the results. Fig.33 As shown in the figure, the positive staining in the image is brown-yellow. Compared with the normal group, a large number of VSMCs in the arterial media in the model group can be seen to be apoptotic. The TPNs and TPNs-siRNA treatment groups can inhibit VSMCs apoptosis, and the therapeutic effect of TPNs-siRNA is more obvious.

[0199] 16. In vivo safety evaluation

[0200] 1 Mouse weight change curve and organ index

[0201] The weight changes of mice in each group were recorded during the administration period. There was no significant difference between the weight of mice in each experimental group and the normal saline group. There was no significant difference in hair, mental state, eating, excrement and activity behavior among the four groups of mice. When the mice in each group were euthanized, in addition to collecting the abdominal aorta, the heart, liver, spleen, lung and kidney of each group of mice were also collected, and the organ index of each major organ was calculated. The results showed that after two weeks of administration, there was no significant difference in the organ index of each group of mice, indicating that TPNs-siRNA and TPNs are highly safe for major organs. Only the model group mice showed a decrease in the liver, which may be due to the AAA disease state.

[0202] 2 In vitro hemolysis assay

[0203] Select a healthy C57 mouse (6-8 weeks), remove the eyeball and collect about 0.5 ml of blood. Put the blood sample in a sodium citrate anticoagulant EP tube and dilute it with PBS into a 2% red blood cell suspension for later use. Mix 0-400 μg / mL TPNs-siRNA and TPNs with 2% red blood cell suspension in equal volumes in a clean EP tube, incubate at 37°C for 3 hours, and observe hemolysis and agglutination reactions. Centrifuge at 1000 rpm for 10 minutes, and detect the absorbance (OD value) of the supernatant at 576 nm of each group. Ultrapure water was used as a positive control, and PBS was used as a negative control. Calculate according to the hemolysis rate (HR, %) = (sample OD-negative OD) / (positive OD-negative OD) × 100%.

[0204] Fig.34 As shown in A, after centrifugation of the H2O positive control group, the upper liquid was bright red, which was caused by the rupture of red blood cells and severe hemolysis. The supernatant of the PBS negative control group was colorless, and there were cell precipitates at the bottom of the tube. In the TPNs-siRNA group, no obvious hemolysis was found, and the hemolysis rate of all concentrations was less than 5%. The same experimental phenomenon also occurred in the vector TPNs group ( Fig.34 B), indicating that TPNs-siRNA and TPNs would not cause aggregation and hemolysis of erythrocytes in vivo, and the two nanoformulations were safe for in vivo injection.

[0205] 3. Hematological analysis

[0206] Blood samples of mice in each group were collected before euthanasia, and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (BUN) and creatinine (CR) in the blood of mice were measured using the corresponding detection kits. The results showed that compared with the normal control group, there was no significant change in the ALT, AST, BUN and CR values ​​of each group, and only TPNs-siRNA showed some differences in the urea level. Overall, the two nanoformulations hardly caused damage to the liver and kidney functions of mice.

[0207] 4 Histological analysis of various organs

[0208] The collected mouse organs were fixed with 4% paraformaldehyde, paraffin sections were made and H&E staining was performed. The H&E staining results of the main organs of the four groups of mice showed that all organs were relatively normal, with almost no pathological changes, indicating that TPNs-siRNA and TPNs have high safety in vivo.

Claims

1. A MMPs inhibitor, characterized in that: It includes siRNA for silencing MMP-2 and siRNA for silencing MMP-9, wherein the siRNA for silencing MMP-2 is any one of siMMP-2-1, siMMP-2-2 and siMMP-2-3, and the siRNA for silencing MMP-9 is any one of siMMP-9-1, siMMP-9-2 and siMMP-9-3; The nucleic acid sequence of the siMMP-2-1 is shown in SEQ ID NO: 1 and SEQ ID NO: 2; The nucleic acid sequence of the siMMP-2-2 is shown in SEQ ID NO: 3 and SEQ ID NO: 4; The nucleic acid sequence of the siMMP-2-3 is shown in SEQ ID NO: 5 and SEQ ID NO: 6; The nucleic acid sequence of the siMMP-9-1 is shown in SEQ ID NO: 7 and SEQ ID NO: 8; The nucleic acid sequence of the siMMP-9-2 is shown in SEQ ID NO: 9 and SEQ ID NO: 10; The nucleic acid sequences of the siMMP-9-3 are shown in SEQ ID NO:11 and SEQ ID NO:

12.

2. The MMPs inhibitor according to claim 1, characterized in that: Including siMMP-2-1 and siMMP-9-3.

3. Use of the MMPs inhibitor according to claim 1 or 2 in the preparation of nanoformulations for targeted delivery of therapeutic drugs.

4. The use according to claim 3, characterized in that: The therapeutic drugs include but are not limited to drugs or preparations for treating abdominal aortic aneurysm, atherosclerosis or anti-skin photoaging.

5. The use according to claim 3, characterized in that: The nano preparation carries the matrix metalloproteinase MMPs inhibitor on a delivery carrier, and the delivery carrier is a TPNs carrier.

6. The use according to claim 5, characterized in that: The nanoformulation is prepared by the following preparation method: after adding EGCG solution to HEPES buffer at pH 8.0, the same volume of siMMP-2-1 and siMMP-9-3 solutions are respectively added and pre-stirred for 30 minutes, and then MnCl2 solution is slowly added dropwise, vigorously stirred for 2.5 hours at a constant temperature of 37°C, the reaction solution is taken, centrifuged, the supernatant is discarded, and the solution is washed with HEPES buffer and resuspended to obtain the TPNs-siRNA nanoformulation.

7. The application of MMPs inhibitors and TPNs synergistically in inhibiting cellular oxidative stress.

8. Application of the synergistic effect of MMPs inhibitors and TPNs in improving the targeting of therapeutic drug delivery in vivo.

9. The use according to claim 8, characterized in that: The therapeutic drugs include but are not limited to drugs or preparations for treating abdominal aortic aneurysm, atherosclerosis or anti-skin photoaging.

10. The use according to claim 8 or 9, characterized in that: The abdominal aortic aneurysm treatment drug is a nano preparation in which a MMPs inhibitor is carried on a TPNs carrier.

Images