Nanoparticles for targeting fibroblasts to deliver miR-92a-1-5p as well as preparation method and application of nanoparticles

Abstract

The invention discloses a nano-particle for delivering miR-92a-1-5p by targeting fibroblasts as well as a preparation method and application of the nano-particle. According to the preparation method, a poly (ethylene glycol methacrylate)-polylysine nano material with ROS reactivity is adopted, PML nano particles are prepared through a cross-linking reaction, dialysis purification, ultrasonic treatment and centrifugation, the PMLP nano particles are connected with a collagen fiber aggregation peptide fragment CREKA in a covalent coupling mode, and the fibroblast targeting nano particles with PML as a core and CREKA as surface modification are formed. The miR-92a-1-5 nucleic acid small molecule drug is embedded in the core hydrophobic cavity of the PMLP nano particle; the prepared nano-particles can realize targeted delivery of fibroblasts in fibrotic diseases and release miR-92a-1-5p nucleic acid small molecules wrapped in the nano-particles, so that a specific anti-fibrosis treatment effect is finally achieved, and the nano-particles have a good application prospect.

Description

Technical Field

[0001] The invention relates to a nanoparticle for delivering miR-92a-1-5p by targeting fibroblasts and a preparation method and application thereof, belonging to the technical field of drug delivery. Background Art

[0002] Today, the morbidity and mortality of cardiovascular diseases (CVDs) remain high worldwide. Many end-stage cardiovascular diseases such as myocardial infarction, hypertrophic cardiomyopathy, dilated cardiomyopathy, diabetic cardiomyopathy, aortic valve stenosis, etc. can lead to pathological remodeling of the myocardium, increase matrix stiffness, and ultimately lead to myocardial fibrosis. Myocardial fibrosis, as a myocardial scar event, can be divided into perivascular, focal, replacement, or interstitial fibrosis according to different pathological characteristics and clinical manifestations. The latter includes reactive fibrosis and infiltrative fibrosis. Fibrotic remodeling of the myocardium can limit ventricular systolic and diastolic function and induce heart failure. Currently, the effective clinical treatment for myocardial infarction is timely and effective revascularization, including coronary artery bypass grafting (CABG), percutaneous coronary intervention (PCI), and drug thrombolytic therapy. However, reperfusion injury caused by revascularization has a certain degree of damage to myocardial cells and induces myocardial cell apoptosis. Therefore, it is necessary to explore new strategies to prevent fibrosis or alleviate fibrosis in the treatment of myocardial infarction. Based on the key targets of the fibrosis pathway after myocardial infarction, targeted delivery of anti-fibrotic drugs or bioactive factors to inhibit fibrosis-related molecules such as TGF-β, inhibit the transformation of fibroblasts in the myocardium into myofibroblasts, and avoid excessive collagen production and deposition is a new strategy.

[0003] In recent years, studies have found that microRNA (miRNA) can recognize target mRNA through complementary base pairing, guide its degradation or inhibit its translation, and play an important role in the occurrence and development of various diseases including cardiovascular diseases. It also has a certain regulatory and improvement effect in cardiac pathological conditions such as myocardial fibrosis. Although direct administration of exogenous miRNA can improve cardiac function after myocardial infarction, the degradation rate of direct delivery of miRNA is high, the retention time in the infarct area is short, and the efficiency of target cell endocytosis is also low. Subsequently, adeno-associated virus (AAV) vectors were developed for the delivery of miRNA, but AAV vectors still have their own limitations, such as potential immunogenicity, high cytotoxicity, and short half-life in blood and serum. Fortunately, with the development of nanotechnology, non-viral vectors have gradually been used, including liposomes, inorganic nanoparticles, and polymeric nanoparticles. They have low immunogenicity and long half-life. The chemical or biological modification of their surface can also give nanoparticles targeting characteristics, assisting them to load small molecule drugs, cytokines or nucleic acids and home to specific tissue cells to achieve precision treatment.

[0004] In our previous research, we found a key molecule miR-92a-1-5p that can inhibit fibroblast activation and preliminarily analyzed its molecular mechanism of inhibiting fibrosis. However, direct delivery of miRNA has problems such as endocytosis, off-target effects, and short half-life. Nanomaterials, such as liposomes, polymers (PLGA), inorganic nanoparticles (AuNPs, MnO2, etc.), natural nanoparticles (HDL, HA) and biomimetic nanoparticles (cell membrane coatings), can load "drugs" such as chemicals, proteins, peptides and nucleic acids and deliver them to the damaged area by virtue of their physicochemical properties and surface modification potential, which greatly improves the shortcomings of miRNA naked drugs such as short half-life and low bioavailability in vivo. When the heart is hit by ischemia and hypoxia, the explosive production of oxygen free radicals in a short period of time will cause acute and chronic damage to cells, while ROS-responsive nanomaterials can selectively deliver and release drugs to lesion targets in oxidative microenvironments, and have good delivery efficiency for ROS-accumulated damaged tissues such as ischemia or reperfusion injury. Therefore, it is necessary to develop a ROS-responsive nanoparticle delivery system to achieve targeted delivery to fibroblasts by relying on fibroblast-targeting peptides to release miR-92a-1-5p to achieve anti-fibrotic therapeutic effects. Summary of the invention

[0005] The present invention aims to provide a nanoparticle for targeted fibroblast delivery of miR-92a-1-5p and a preparation method and application thereof, which can achieve targeted fibroblast delivery in fibrotic diseases and release the miR-92a-1-5p nucleic acid small molecules encapsulated therein, ultimately achieving a specific anti-fibrosis therapeutic effect.

[0006] In order to achieve the above-mentioned purpose, the present invention provides a nanoparticle for delivering miR-92a-1-5p to targeted fibroblasts, wherein the nanoparticle is a PMLP nanoparticle encapsulated with a small molecule nucleic acid drug miR-92a-1-5, wherein the PMLP nanoparticle is prepared from poly(ethylene methacrylate) and polylysine by cross-linking reaction, dialysis purification, ultrasonic treatment and centrifugation to prepare PML nanoparticles, and then the PMLP nanoparticle is connected to a collagen fiber aggregation peptide segment CREKA by covalent coupling to form a fibroblast-targeted nanoparticle PMEMA-polylysine-peptide with PML as the core and CREKA as the surface modification, i.e., PMLP nanoparticle; the miR-92a-1-5 is encapsulated in the core hydrophobic cavity of the PMLP nanoparticle.

[0007] The present invention also provides a method for preparing the above-mentioned nanoparticles, comprising the following steps:

[0008] Step 1: Dissolve PMEMA and Polylysine in organic solvents respectively, mix them thoroughly and then mix them to obtain a mixed solution;

[0009] Step 2: Add cross-linking agents EDC and NHS to the mixed solution obtained in step 1 and stir to react;

[0010] Step 3: obtaining a PML nanoparticle suspension by dialysis purification, sonication, and centrifugation;

[0011] Step 4: covalently couple the PML nanoparticle suspension with the CREKA peptide segment, and obtain a suspension of PMLP nanoparticles modified with the CREKA peptide segment surface after purification;

[0012] Step 5: Mix the PMLP nanoparticle suspension with miR-92a-1-5p, vortex and then let stand to obtain PMLP nanoparticles loaded with miR-92a-1-5p nucleic acid small molecule drugs.

[0013] In some embodiments of the present invention, the organic solvent in step 1 is anhydrous ethanol.

[0014] Preferably, the preparation of the suspension of PMLP nanoparticles in step 4 specifically comprises the following steps:

[0015] Step S1: adding a carboxylation reagent to the PML nanoparticle suspension, and using the carboxylation reagent to introduce carboxyl groups on the surface of the PML nanoparticles, so as to facilitate the subsequent covalent binding of the nanoparticle surface with the peptide segment;

[0016] Step S2: dissolving the CREKA peptide in PBS buffer, adding maleimide-PEG-NHS, and stirring the reaction at room temperature to activate the thiol group at the end of the peptide (the thiol group on Cys);

[0017] Step S3: Add the CREKA peptide solution activated in step S2 to the PML nanoparticle suspension, stir the reaction to complete the covalent coupling reaction; and dialysis purification is performed to obtain the product.

[0018] In some embodiments of the present invention, the carboxylation reagent in step S1 is succinic anhydride.

[0019] Preferably, the pH value of the PBS buffer in step S2 is 6.5-7.5.

[0020] Preferably, the stirring reaction in step S3 is carried out under the conditions of stirring the reaction at room temperature for 10-15 hours.

[0021] Preferably, the conditions for the dialysis purification in step S3 are: dialysis in PBS buffer for 24-48 hours to remove unreacted peptides and cross-linking agents.

[0022] Preferably, in step 1, the final concentrations of PMEMA and Polylysine dissolved in organic solvents are controlled within the range of 1-5 mg / mL.

[0023] Preferably, the molar ratio of EDC to NHS in step 2 is 1:1-2; the stirring reaction conditions are: stirring the reaction at room temperature for 20-30 hours;

[0024] Preferably, the dialysis purification in step 3 comprises: dialysis in deionized water for 24-48 hours, changing the water every 6-8 hours to remove unreacted reagents and organic solvents; the ultrasonic treatment condition is 100-200W power for 5-15min; the centrifugation condition is: centrifugation at 8000-12,000rpm for 5-15min.

[0025] The present invention also provides a use of the above-mentioned nanoparticles, wherein the use is selected from any one of the following A1-A8:

[0026] A1. A drug delivery system for preparing a drug delivery system for targeting fibroblasts and releasing miR-92a-1-5p;

[0027] A2. Preparation of nano-delivery systems for achieving ROS responsiveness;

[0028] A3. Preparation of a nano-delivery system for targeting fibroblasts and high expression of miR-92a-1-5p;

[0029] A4. Preparation of nanodelivery systems for improving fibroblast fiber activation;

[0030] A5. Preparation of a nanodelivery system for inhibiting fibroblast migration ability;

[0031] A6. Preparation of a nanodelivery system for inhibiting fibroblast collagen contraction;

[0032] A7. Preparation of cell models for screening therapeutic drugs for fibrotic diseases;

[0033] A8. Preparation of nano-delivery system for improving the efficacy of miR-92a-1-5p;

[0034] A9. Used for preparing medicines for treating fibrotic diseases.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention cross-links PMEMA and Polylysine to form PML nanoparticles, which have ROS responsiveness and can actively respond to ROS signals in the ischemic and hypoxic microenvironment of ischemic diseases such as myocardial infarction to achieve rapid tracking of the delivery system and drug release; the present invention covalently couples PML nanoparticles with CREKA peptide segments to prepare PMLP nanoparticles, realizes the covalent coupling modification of fibroblast targeting peptides on the surface of nanoparticles, and helps to achieve specific and precise targeted delivery of scar fibers and fibroblasts; the targeted delivery nanoparticles designed by the present invention well encapsulate anti-fibrosis nucleic acid small molecule drugs, and solve the problems of short half-life and low bioavailability of nucleic acid drugs in disease treatment; the preparation method of the present invention is non-toxic, environmentally friendly, economical and efficient, and has high clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The particle size and surface charge of PML and PMLP nanoparticles were determined for dynamic light scattering.

[0038] Figure 2 Transmission electron microscopy was used to observe the morphological characteristics of PML and PMLP nanoparticles.

[0039] Figure 3 To detect the internalization of PMLP@miR-92a-1-5p nanoparticles in fibroblasts in response to H2O2 treatment by flow cytometry;

[0040] Figure 4 Immunofluorescence and western blot were used to detect that PMLP@miR-92a-1-5p nanosystem mediated fibroblast internalization of nanoparticles and achieved high expression of miR-92a-1-5p;

[0041] Figure 5 The effect of PMLP@miR-92a-1-5p nanosystem on fibroblast activation under TGF-β-induced fibrosis intervention conditions;

[0042] Figure 6 The effect of PMLP@miR-92a-1-5p nanosystem on fibroblast migration under TGF-β-induced fibrosis intervention conditions;

[0043] Figure 7 This is the effect of PMLP@miR-92a-1-5p nanosystem on collagen contraction of fibroblasts under TGF-β-induced fibrosis intervention conditions.

[0044] Figure 8 The PMLP@miR-92a-1-5p nanosystem is targeted to deliver cardiac fibroblasts after myocardial infarction to improve the effect of fibrosis. DETAILED DESCRIPTION

[0045] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0046] Example 1: Synthesis of PMLP nanoparticles loaded with miR-92a-1-5p and targeting fibroblasts

[0047] 1. Synthesis of PMEMA-polylysine (PML) nanoparticles

[0048] (1) Solution preparation

[0049] PMEMA (polyethylene methacrylate) and Polylysine (polylysine) were dissolved in anhydrous ethanol respectively, so that the concentration of the two was controlled within the range of 1-5 mg / mL, and stirred with a magnetic stirrer until PMEMA and Polylysine were completely dissolved.

[0050] (2) Cross-linking reaction

[0051] PMEMA and Polylysine solution were mixed in a ratio of 1:1, and cross-linking agent EDC / NHS was added so that the molar ratio of EDC to NHS was controlled at 1:1 to 1:2. The mixture was stirred and reacted at room temperature for 24 hours.

[0052] (1) Dialysis purification

[0053] The mixture reacted for 24 hours was transferred to a dialysis bag and dialyzed in deionized water for 48 hours, with the water being changed every 6-8 hours to remove unreacted reagents and organic solvents.

[0054] (4) Ultrasonic treatment and centrifugation

[0055] The dialyzed solution was sonicated at 100 W for 10 min to disperse the nanoparticles, and centrifuged at 10,000 rpm for 10 min to remove large particles and undispersed substances.

[0056] 2. PML nanoparticles covalently coupled to CREKA peptide to prepare PMLP peptide

[0057] (1) PML nanoparticle pretreatment

[0058] A carboxylating agent (succinic anhydride) was used to introduce carboxyl groups on the surface of the nanoparticles, so that the PML nanoparticles have surface active groups.

[0059] (2) CREKA peptide activation

[0060] Cross-linking reaction using maleimide-PEG-NHS: The CREKA peptide was dissolved in PBS buffer (pH 6.5-7.5), and then maleimide-PEG-NHS was added and stirred at room temperature for 1 hour to activate the thiol (Cys) at the terminal of the peptide.

[0061] (3) Covalent coupling reaction

[0062] The activated CREKA peptide solution was added to the PML nanoparticle suspension and stirred at room temperature for 12 hours.

[0063] (4) Purification reaction

[0064] The reaction mixture was transferred to a dialysis bag and dialyzed in PBS buffer for 24-48 hours to remove unreacted peptides and cross-linkers. The mixture was then centrifuged at 10,000 rpm for 10 minutes to remove unbound peptides. The mixture was then freeze-dried to obtain PMLP nanoparticle powder.

[0065] 3. PMLP nanoparticles loaded with miR-92a-1-5p to prepare PMLP@miR-92a-1-5p nanoparticle system

[0066] (1) Dissolve PMLP powder in DMSO to prepare a 20 mg / mL PML nanoparticle stock solution.

[0067] (2) The PMLP nanoparticle stock solution was added dropwise into the vigorously stirred acetate buffer (pH 5.25) so that the volume ratio of the stock solution to the buffer was controlled at 1 / 10;

[0068] (3) Add an equal volume of miRNA solution to control the PMLP / miRNA molar concentration mass ratio to 1 / 2, 1 / 1, 2 / 1, 5 / 1, 10 / 1, 20 / 1, and 50 / 1. Vortex for 30 seconds and let stand for 30 minutes to obtain the PMLP@miR-92a-1-5p nanoparticle system.

[0069] 4. Determination of PML and PMLP Nanoparticle Size and Surface Charge Using Dynamic Light Scattering

[0070] (1) PML / PMLP nanoparticle size determination: Dilute the PML / PMLP nanoparticle suspension with buffer to an appropriate concentration, and use an ultrasonic instrument to disperse the aggregated nanoparticle clumps. Turn on the dynamic light scattering instrument, preheat and calibrate the instrument. Add the diluted PML / PMLP nanoparticle suspension to a dedicated measurement cell, and avoid generating bubbles. Set the temperature to 25°C, the measurement angle to 90°, the measurement time and number to 10-30 seconds / time, and repeat 3-5 times. Start the measurement and record the particle size distribution results (such as average particle size, polydispersity index PDI, etc.).

[0071] (2) Determination of the surface charge of PML / PMLP nanoparticles: Add the diluted PML / PMLP nanoparticle suspension into a dedicated Zeta potential measurement cell. Set the temperature to 25°C, the electric field strength to the recommended value of the instrument, and the number of measurements to 3-5 times. Start the measurement and record the Zeta potential value.

[0072] (3) Results: The test results are as follows: Figure 1 As shown. The particle size measurement results show that the average particle size of PML nanoparticles is 84.93nm, while PMLP has a larger average particle size of 121.2nm due to the surface modification of CREKA peptide. After loading with miR-92a-1-5p nucleic acid small molecule drugs, both PML and PMLP showed an increase in the volume of nanoparticles, and their average particle sizes reached 131.8nm and 137.8nm, respectively. The surface charge measurement results show that the average surface charge of PML nanoparticles is 21.03mV, while PMLP has an average surface charge reduced to 12.37mV due to the surface modification of negatively charged CREKA peptide. After loading with negatively charged miR-92a-1-5p nucleic acid small molecule drugs, both PML and PMLP have a decrease in average surface charge, and their average surface charges are 15.53nm and 10.67nm, respectively. The above results indicate the successful preparation of PML and PMLP, and that both of them successfully packaged the miR-92a-1-5p small molecule nucleic acid drug.

[0073] Example 2: Morphological characteristics of PML and PMLP nanoparticles observed by transmission electron microscopy

[0074] 1. Sample Preparation

[0075] In order to observe the morphological characteristics of PML and PMLP nanoparticles, as well as their morphological changes in response to ROS stress in the ischemic hypoxic microenvironment, the PML / PMLP nanoparticle suspension was diluted to a concentration of 0.1 mg / mL with buffer and buffer added with H2O2, and ultrasonic treatment was performed using an ultrasonicator to disperse the agglomerated nanoparticles.

[0076] 2. Copper mesh processing

[0077] Use tweezers to carefully remove a piece of copper mesh support film, avoid touching the surface of the film, and use ultraviolet light to treat the copper mesh for 5-10 minutes.

[0078] 3. Sample Loading

[0079] Use a pipette to take 2 μL of the diluted and ultrasonically treated PML / PMLP nanoparticle suspension, and drop the suspension onto the surface of the copper mesh support membrane. Let it stand for 1 minute to allow the nanoparticles to be completely adsorbed on the copper mesh membrane, and gently absorb excess liquid from the edge of the copper mesh with filter paper.

[0080] 4. Transmission Electron Microscope Observation

[0081] Place the prepared copper mesh into the transmission electron microscope sample holder and insert the sample holder into the transmission electron microscope. After setting the appropriate parameters, gradually adjust from low magnification to high magnification to observe and take pictures of the sample.

[0082] 5. Result: The test result is as follows Figure 2 As shown in the figure, PML and PMLP nanoparticles are evenly distributed, and the particle sizes are not much different. After H2O2 treatment, both PML and PMLP nanoparticles showed destruction and aggregation of nanoparticles.

[0083] Example 3: Flow cytometry detection of fibroblasts endocytosed in response to PMLP@miR-92a-1-5p nanoparticles under H2O2 treatment

[0084] 1. Preparation of PMLP nanoparticles carrying FITC label and packaging of miR-92a-1-5p carrying cy3.3 label

[0085] (1) adding 1 mg / mL FITC to the 20 mg / mL PMLP nanoparticle stock solution prepared in Example 1 and stirring overnight at room temperature in the dark to prepare PMLP nanoparticles PMLP-FITC carrying a FITC label;

[0086] (2) The PMLP nanoparticle stock solution was added dropwise into the vigorously stirred acetate buffer (pH 5.25) so that the volume ratio of the stock solution to the buffer was controlled at 1 / 10;

[0087] (3) Add an equal volume of miR-92a-1-5p-cy3.3 solution to control the molar concentration mass ratio of PMLP / miRNA to 1 / 2, 1 / 1, 2 / 1, 5 / 1, 10 / 1, 20 / 1, and 50 / 1. Vortex for 30 seconds and let stand for 30 minutes to obtain the PMLP-FITC@miR-92a-1-5p-cy3.3 nanoparticle system.

[0088] 2. Fibroblast Preparation and Treatment

[0089] The fibroblast cell line was seeded in a 6-well plate in advance, and after the cells adhered, 0, 25, 50, 75, and 100 μM H2O2 solutions were added to induce ROS production. At the same time, the above-mentioned PMLP-FITC@miR-92a-1-5p-cy3.3 nanoparticles carrying FITC and cy3.3 labels were added in the optimal ratio and treated under 5% CO2, 37°C in the dark for 24 hours.

[0090] 3. Flow cytometry

[0091] (1) Digest the fibroblast cell line treated above with 0.25% trypsin, terminate with complete medium, centrifuge at 1000 rpm for 5 min, adjust the cell density of each tube to 1×10^6 / mL, wash the cells twice with PBS, remove the supernatant by centrifugation, and keep away from light.

[0092] (2) Suspend the fibroblasts in an appropriate flow staining buffer. Turn on the flow cytometer, preheat and calibrate the instrument. Set the fluorescence channels of the flow cytometer: FITC: usually use 488nm laser excitation, detect 530 / 30nm emission light. PE: usually use 488nm laser excitation, detect 575 / 26nm emission light. Use unstained cells or single-stained cells to adjust compensation. Finally, perform flow cytometry detection and record the fluorescence signal of at least 10,000 cells.

[0093] 4. Results: The flow cytometry results are as follows Figure 3 As shown, as the H2O2 concentration increases in a gradient, the FITC and cy3.3 signals in the fibroblasts gradually increase, indicating that with the accumulation of ROS in the fibroblasts, PMLP loaded with miR-92-1-5p is more easily phagocytosed by fibroblasts due to its ROS tropism.

[0094] Example 4: Fibroblasts internalize PMLP nanoparticles and detect high expression of miR-92a-1-5p in fibroblasts

[0095] 1. Preparation of PMLP nanoparticles carrying FITC label and packaging of miR-92a-1-5p carrying cy3.3 label

[0096] (1) adding 1 mg / mL FITC to the 20 mg / mL PMLP nanoparticle stock solution prepared in Example 1 and stirring overnight at room temperature in the dark to prepare PMLP nanoparticles PMLP-FITC carrying a FITC label;

[0097] (2) The PMLP nanoparticle stock solution was added dropwise into the vigorously stirred acetate buffer (pH 5.25) so that the volume ratio of the stock solution to the buffer was controlled at 1 / 10;

[0098] (3) Add an equal volume of miR-92a-1-5p-cy3.3 solution to control the molar concentration mass ratio of PMLP / miRNA to 1 / 2, 1 / 1, 2 / 1, 5 / 1, 10 / 1, 20 / 1, and 50 / 1. Vortex for 30 seconds and let stand for 30 minutes to obtain the PMLP-FITC@miR-92a-1-5p-cy3.3 nanoparticle system.

[0099] 2. Fibroblast Preparation and Treatment

[0100] The fibroblast cell line was seeded in a 6-well plate and confocal dish in advance. After the cells adhered to the wall, the PMLP-FITC@miR-92a-1-5p-cy3.3 nanoparticles carrying FITC and cy3.3 labels were added at different concentrations and cultured in the dark under 5% CO2 and 37°C conditions for 24 h.

[0101] 3. Cell Immunofluorescence Sample Processing and Imaging

[0102] After culturing for 24 hours, the fibroblasts were washed twice with PBS buffer in the dark, and then Hoechst live cell nuclear staining solution was added. After incubation for 30 minutes, they were washed three times with PBS buffer, and confocal microscopy was used to perform fluorescence imaging of fibroblasts.

[0103] 4. Obtaining cDNA and identifying the overexpression effect of miR-92a-1-5p by RT-qPCR

[0104] (1) RNA extraction kit provided by Shanghai Sangon Biotechnology Co., Ltd. was used to extract RNA from fibroblasts treated with PMLP-FITC@miR-92a-1-5p-cy3.3 nanoparticles at different concentrations. Bulge-LoopTM miRNART Primer (20 μM) provided by Ruibo Biotechnology Co., Ltd. was added with an appropriate amount of RNase-free H2O to prepare Bulge-LoopTM miRNART Primer (5 μM). Finally, 10 μl of RT reaction system was mixed and centrifuged instantly. The reverse transcription reaction program was designed as follows: 42°C for 60 min, 70°C for 10 min, and finally cDNA was obtained by reverse transcription.

[0105] (2) RT-qPCR identification of miR-92a-1-5p overexpression effect

[0106] The RT-qPCR reaction system was prepared with a total volume of 20 μl, consisting of 10 μl of SYBR Green Mix (Ruibo Biotechnology Co., Ltd.) containing Taq enzyme, dNTP mix, PCR Buffer, SYBR Green I, etc., 0.6 μl of upstream primer, 0.6 μl of downstream primer, 8 μl of nuclease-free water, and 0.8 μl of cDNA template obtained in step 1. The prepared reaction system was placed on a fluorescent quantitative PCR instrument for RT-qPCR amplification reaction to detect the relative expression of miR-92a-1-5p in fibroblasts (with U6 gene as the internal reference gene). The relative expression of miR-92a-1-5p was calculated using the 2-ΔΔCT method.

[0107] The upstream primer for amplifying the miR-92a-1-5p gene was: 5'-AGGTTGGGATCGGTTGCAAT-3, and the downstream primer was 5'-AGCATTGCAACCGATCCCAA-3'.

[0108] The upstream primer for amplifying the U6 gene was 5′-CTCGCTTCGGCAGCACA-3′, and the downstream primer was 5′-ACGCTTCACGAATTTGCGT-3′.

[0109] 5. Result: The test result is as follows Figure 4 As shown, immunofluorescence imaging indicates that PMLP carrying FITC label is successfully internalized by fibroblasts, and miR-92a-1-5p-cy3.3 encapsulated in PMLP is also successfully engulfed by fibroblasts. In addition, RT-qPCR results indicate that with the increase of the concentration of co-incubated PMLP-FITC@miR-92a-1-5p-cy3.3 nanoparticles, the expression level of miR-92a-1-5p in fibroblasts is gradually increased. The above results indicate that fibroblasts can engulf the PMLP@miR-92a-1-5p nanoparticle system, and the engulfed miR-92a-1-5p can be released intracellularly to exert its effect, thereby achieving high expression of miR-92a-1-5p in fibroblasts.

[0110] Example 5: Effect of PMLP@miR-92a-1-5p nanosystem on fibroblast fiber activation under TGF-β-induced fibrosis intervention conditions;

[0111] 1. Modeling of fibroblast activation induced by TGF-β recombinant protein

[0112] The naked PMLP nanoparticles and PMLP@miR-92a-1-5p nanoparticle system constructed in Example 1 were used to intervene in fibroblasts seeded in a 6-well plate, and 20 ng / mL of TGF-β recombinant protein was added to induce fiber activation. The cells were treated under culture conditions of 5% CO2 and 37°C for 24 h.

[0113] 2. Western blot experiment:

[0114] Total cellular protein from fibroblasts was extracted using neutral lysis buffer RIPA, and after denaturation, SDS-PAGE electrophoresis was performed at 100V for 90min; transfer to membrane at 300mA for 100min; PVDF membrane was blocked with fast blocking buffer for 30min; 1000:1 diluted β-actin rabbit primary antibody (Cell Signaling Technology, USA), 1000:1 diluted MMP9 rabbit primary antibody (Cell Signaling Technology, USA), 1000:1 diluted FSP-1 rabbit primary antibody (Cell Signaling Technology, USA), 1000:1 diluted Vimentin rabbit primary antibody (Cell Signaling Technology, USA), 1000:1 diluted α-SMA rabbit primary antibody (Abcam, UK) were incubated overnight at 4℃; the primary antibody was recovered and the membrane was washed 3 times with 1×TBST solution on a shaker for 10min each time; 5000:1 diluted mouse II (Cell Signaling Technology, USA) was incubated at room temperature for 1h; and developed.

[0115] 3. Experimental results

[0116] The results of Western blot development are as follows Figure 5 As shown. Under the induction of TGF-β, the expression levels of MMP9, Vimentin, α-SMA and FSP-1 in fibroblasts increased significantly, indicating that TGF-β successfully induced fibroblast activation. The PMLP@miR-92a-1-5p nanoparticle system significantly inhibited the expression levels of these proteins, indicating that the PMLP@miR-92a-1-5p nanoparticle system can significantly improve the ability of TGF-β-induced fibroblast activation under fibrotic intervention conditions.

[0117] Example 6: Effect of PMLP@miR-92a-1-5p nanosystem on fibroblast migration under TGF-β-induced fibrosis intervention conditions

[0118] 1. Transwell experiment

[0119] The fibroblast cell line was digested with 0.25% trypsin, and centrifuged at 1000rpm for 5min after termination with complete medium; the cells were inoculated in the upper chamber of the Transwell chamber (Beijing Lanjieke Technology Co., Ltd.) with 20,000 cells per well and 200μl serum-free medium, and the naked PMLP nanoparticles and PMLP@miR-92a-1-5p nanoparticles constructed in Example 1 were added to the upper chamber at the optimal concentration ratio. 500μl of high-glucose DMEM medium containing 10% fetal bovine serum was added to the lower chamber of the transwell chamber, and TGF-β recombinant protein was added to the lower chamber to a final concentration of 20ng / μl to induce fiber activation. The culture plate was placed in an incubator at 37°C and 5% CO2 for 24h; fixed with 4% PFA at room temperature for 30 minutes, and stained with 0.1% crystal violet at room temperature for 10 minutes; image acquisition.

[0120] 2. Experimental results

[0121] Transwell migration and statistical analysis Figure 6 The results showed that the PMLP@miR-92a-1-5p nanoparticle system could significantly inhibit TGF-β-induced fibroblast migration.

[0122] Example 7: Effect of PMLP@miR-92a-1-5p nanosystem on collagen contraction of fibroblasts under TGF-β-induced fibrosis intervention conditions.

[0123] 1. Preparation of Cell Suspension

[0124] The fibroblasts were digested with 0.25% trypsin, and after termination with complete medium, the cells were centrifuged at 1000 rpm for 5 min. The fibroblasts were resuspended with high-glucose DMEM medium to a density of 400,000 cells / mL, and the suspension was placed on ice for later use.

[0125] 2. Preparation of collagen containing fibroblasts

[0126] To 200 μL of type I rat tail collagen (5 mg / ml), 12 μL of 0.1 mol / 1 NaOH and 23 μL of 10×DMEM medium containing phenol red were added in sequence and mixed immediately. Then 760 μL of the above-mentioned cell suspension placed on ice was added, and the cell suspension and collagen were fully mixed and immediately added to a 24-well plate, 300 μL per well, to avoid the generation of bubbles. The well plate was placed at 37°C for 1 hour until the gel solidified, and 500 μL of high-glucose DMEM containing 20 ng / μL of TGF-β was added to each well to induce fibroblast activation and collagen contraction. At the same time, the naked PMLP nanoparticles and PMLP@miR-92a-1-5p nanoparticles constructed in Example 1 were added to the culture medium according to the optimal concentration ratio and incubated together.

[0127] 3. Imaging and Analysis of Collagen Contraction

[0128] After 24 h of TGF-β-induced fiber activation and collagen contraction, the well plate was placed under a developer and imaged in white light mode to measure the collagen area and calculate the collagen contraction rate.

[0129] 4. Experimental Results

[0130] The results of the collagen contraction experiment are as follows Figure 7 The results showed that the PMLP@miR-92a-1-5p nanoparticle system could significantly inhibit TGF-β-induced fibroblast collagen contraction.

[0131] The nanoparticles prepared by the present invention that can deliver miR-92a-1-5p to targeted fibroblasts can achieve targeted delivery to fibroblasts in fibrotic diseases and release the miR-92a-1-5p nucleic acid small molecules encapsulated therein, ultimately achieving a specific anti-fibrosis therapeutic effect, solving the problems of short half-life and poor bioavailability of nucleic acid small molecule drugs, and laying a foundation for in-depth research on the molecular mechanism of miR-92a-1-5p in inhibiting fibrosis and the application of miR-92a-1-5p in the treatment of fibrotic diseases.

[0132] Example 7: PMLP@miR-92a-1-5p nanosystem targeted delivery of cardiac fibroblasts after myocardial infarction to improve myocardial fibrosis

[0133] 1. Establishment of Mouse Myocardial Infarction Model

[0134] C57 male mice aged 6-8 weeks were selected to establish a myocardial infarction model. The mice were anesthetized with 2% isoflurane by inhalation using an isoflurane infusion system. A 0.5-1 cm incision was made at the intercostal area below the axilla on the left chest of the mouse, and external force was applied to expose part of the heart to the outside of the body. A suture needle and 8-0 suture were passed through the heart about 2-3 mm below the tip of the left atrial appendage to achieve permanent ligation of the middle and distal segments of the anterior descending branch of the left coronary artery of the mouse heart, and then the heart was reset to its original anatomical position in the body. The sham operation group was given the same incision operation, but the coronary artery was not ligated.

[0135] 2. Tail vein injection of PMLP@miR-92a-1-5p nanosystem

[0136] After myocardial infarction modeling in mice, 100 μL of normal saline was immediately injected into the tail vein of mice in the sham operation group and the myocardial infarction group, and 100 μL of PMLP@miR-92a-1-5p nanoparticle solution dissolved in normal saline was injected into the tail vein of mice in the PMLP@miR-92a-1-5p treatment group.

[0137] 3.Masson staining

[0138] 28 days after myocardial infarction and tail vein injection of PMLP@miR-92a-1-5p nanosystem in mice, the mouse hearts were fixed in 4% paraformaldehyde and then paraffin sections were prepared. After dewaxing with xylene and rehydration with gradient ethanol and deionized water, iron hematoxylin staining, acid fuchsin staining, phosphomolybdic acid / phosphotungstic acid differentiation and aniline blue staining were performed. Finally, dehydration, transparency and sealing were performed. The degree of fibrosis in the heart sections was observed under a microscope.

[0139] 4. Experimental Results

[0140] Masson staining results of cardiac fibrosis Figure 8 The results showed that significant myocardial fibrosis could be caused one month after myocardial infarction, and the PMLP@miR-92a-1-5p nanosystem was effectively inhibited by targeted delivery of cardiac fibroblasts via tail vein injection.

[0141] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A nanoparticle for delivering miR-92a-1-5p to fibroblasts, characterized in that: The nanoparticles are PMLP nanoparticles embedded with the nucleic acid small molecule drug miR-92a-1-5. The PMLP nanoparticles are made of poly(ethylene methacrylate) and polylysine, and are prepared by cross-linking reaction, dialysis purification, ultrasonic treatment and centrifugation to prepare PML nanoparticles. The PMLP nanoparticles are then connected to the collagen fiber aggregation peptide segment CREKA by covalent coupling to form PMEMA-polylysine-peptide, a fibroblast-targeted nanoparticle with PML as the core and CREKA as the surface modification, i.e., PMLP nanoparticles. The miR-92a-1-5 is embedded in the core hydrophobic cavity of the PMLP nanoparticles.

2. The method for preparing nanoparticles according to claim 1, characterized in that: The following steps are involved: Step 1: Dissolve PMEMA and Polylysine in organic solvents respectively, mix them thoroughly and then mix them to obtain a mixed solution; Step 2: Add cross-linking agents EDC and NHS to the mixed solution obtained in step 1 and stir to react; Step 3: obtaining a PML nanoparticle suspension by dialysis purification, sonication, and centrifugation; Step 4: covalently couple the PML nanoparticle suspension with the CREKA peptide segment, and obtain a suspension of PMLP nanoparticles modified with the CREKA peptide segment surface after purification; Step 5: Mix the PMLP nanoparticle suspension with miR-92a-1-5p, vortex and then let stand to obtain PMLP nanoparticles loaded with miR-92a-1-5p nucleic acid small molecule drugs.

3. The preparation method according to claim 2, characterized in that: The preparation of the suspension of PMLP nanoparticles in step 4 specifically includes the following steps: Step S1: adding a carboxylation reagent to the PML nanoparticle suspension, and using the carboxylation reagent to introduce carboxyl groups on the surface of the PML nanoparticles, so as to facilitate the subsequent covalent binding of the nanoparticle surface with the peptide segment; Step S2: Dissolve the CREKA peptide in PBS buffer, add maleimide-PEG-NHS, and stir the reaction at room temperature to activate the thiol group at the end of the peptide; Step S3: Add the CREKA peptide solution activated in step S2 to the PML nanoparticle suspension, stir the reaction to complete the covalent coupling reaction; and dialysis purification is performed to obtain the product.

4. The preparation method according to claim 2, characterized in that: In the step 1, PMEMA and Polylysine are dissolved in organic solvents respectively, and the final concentrations are controlled within the range of 1-5 mg / mL.

5. The preparation method according to claim 2, characterized in that: The molar ratio of EDC to NHS in step 2 is 1:1-2; the stirring reaction conditions are: stirring the reaction at room temperature for 20-30 hours.

6. The preparation method according to claim 2, characterized in that: The dialysis purification in step 3 includes: dialysis in deionized water for 24-48 hours, changing the water every 6-8 hours to remove unreacted reagents and organic solvents; the ultrasonic treatment condition is 100-200W power for 5-15 minutes; the centrifugation condition is: centrifugation at 8000-12,000rpm for 5-15 minutes.

7. The use of the nanoparticles according to claim 1, characterized in that: The use is selected from any one of the following A1-A9: A1. A drug delivery system for preparing a drug delivery system for targeting fibroblasts and releasing miR-92a-1-5p; A2. Preparation of nano-delivery systems for achieving ROS responsiveness; A3. Preparation of a nano-delivery system for targeting fibroblasts and high expression of miR-92a-1-5p; A4. Preparation of nanodelivery systems for improving fibroblast fiber activation; A5. Preparation of a nanodelivery system for inhibiting fibroblast migration ability; A6. Preparation of a nanodelivery system for inhibiting fibroblast collagen contraction; A7. Preparation of cell models for screening therapeutic drugs for fibrotic diseases; A8. Preparation of nano-delivery system for improving the efficacy of miR-92a-1-5p; A9. Used for preparing medicines for treating fibrotic diseases.

Images