CN117045583A - miRNA microneedle patch based on Zn-MOF nano delivery carrier and preparation method and application thereof - Google Patents
miRNA microneedle patch based on Zn-MOF nano delivery carrier and preparation method and application thereof Download PDFInfo
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- CN117045583A CN117045583A CN202311042662.0A CN202311042662A CN117045583A CN 117045583 A CN117045583 A CN 117045583A CN 202311042662 A CN202311042662 A CN 202311042662A CN 117045583 A CN117045583 A CN 117045583A
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- mirna
- microneedle patch
- microneedle
- mir
- zif
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Abstract
The invention provides a miRNA microneedle patch based on a Zn-MOF nano delivery carrier, and a preparation method and application thereof, and belongs to the technical field of biological medicines. A miRNA microneedle patch based on a Zn-MOF nanodelivery vehicle, comprising a microneedle-shaped matrix and a miRNA-loaded Zn-MOF nanodelivery vehicle embedded in the needle portion of the microneedle in the matrix; miRNAs include miR-30d or a mimetic or agonist of miR-30 d. The Zn-MOF nanoparticle is used as a gene delivery carrier for the first time, so that the curative effect of miRNA in myocardial ischemia reperfusion injury can be effectively improved, and the prepared microneedle patch is continuously administered in situ, has good biocompatibility and can be used for effectively treating myocardial ischemia reperfusion injury. And the miRNA microneedle patch is also loaded with gold nano particles with good conductivity, and has an important curative effect on improving arrhythmia caused by myocardial ischemia.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a miRNA microneedle patch based on a Zn-MOF nano delivery carrier, and a preparation method and application thereof.
Background
Myocardial infarction is one of the leading causes of cardiovascular death. Timely reperfusion is an effective treatment for preventing myocardial cell necrosis after myocardial infarction, but blood reperfusion can accelerate and amplify myocardial ischemia injury, i.e., myocardial ischemia reperfusion injury, by inducing myocardial cell death and fibrosis. Myocardial apoptosis and myocardial fibrosis severely impair cardiac function due to limited cardiac regeneration. Micrornas (mirnas) can regulate complementary messenger RNAs (mrnas) to affect the cardiovascular system, thus showing great potential in heart repair and regeneration. Applicant's previous studies found that in rodent models of ischemic heart failure, overexpression of miR-30d in the heart was associated with beneficial ventricular remodeling, decreased cardiomyocyte apoptosis, and fibrosis. In heart failure patients, low-solubility circulating miR-30d would enhance pathological left ventricular remodeling and worse prognosis, suggesting that exogenous miR-30d delivery has a prospective therapeutic role in myocardial ischemia reperfusion injury treatment.
Since mirnas are easily degraded by various nucleases in serum, to realize clinical application of mirnas, the problems of premature inactivation, low retention rate and the like of mirnas must be solved. Viral vectors, while highly transduction efficient, are limited by simultaneous inflammatory/immunogenic response, high toxicity and low packaging. Although non-viral vectors such as liposomes exist, it is still a major challenge to avoid premature degradation during endocytosis. In addition, mirnas are not suitable for cardiac in situ administration because the residence time of mirnas in the heart is short, greatly reducing the efficacy of mirnas. Intravenous injection is generally used as a systemic administration method, but only a small fraction of miRNA-loaded nanoparticles reach the target tissue due to clearance of the immune system. Prior studies have shown that the proportion of nanoparticles successfully delivered to the tumor site by intravenous injection is less than 1%. Another method of intracardiac injection can promote aggregation of drugs at the targeted site more than intravenous injection. However, since miRNA has a "one-needle multi-target" characteristic, it can act on other tissues simultaneously, which if injected into the left ventricular cavity, miRNA is delivered to other tissues with blood flow, thereby causing toxic side effects on other organs. Thus, for intramyocardial injection, it is important that the miRNA be precisely injected into the myocardium rather than the left ventricular cavity.
Disclosure of Invention
Therefore, the invention aims to provide the miRNA microneedle patch based on the Zn-MOF nano delivery carrier, which not only prevents degradation of the pharmaceutically active molecule miRNA under the delivery of the Zn-MOF nano delivery carrier, but also realizes in-situ delivery of cardiac muscle, avoids entering the heart chamber, and improves the curative effect of miR-30d on relieving myocardial ischemia reperfusion injury.
The invention provides a miRNA microneedle patch based on a Zn-MOF nano delivery carrier, which comprises a microneedle-shaped matrix and a miRNA-loaded Zn-MOF nano delivery carrier embedded in a needle head part of a microneedle in the matrix;
the Zn-MOF nano delivery carrier is combined with miRNA through electrostatic acting force;
the miRNA comprises miR-30d, miR-30d mimic or miR-30d agonist.
Preferably, the Zn-MOF nano delivery carrier is prepared by self-assembling zinc salt and imidazolyl molecules in a solvent.
Preferably, the zinc salt comprises zinc acetate or zinc nitrate;
the imidazolyl molecule comprises 2-methylimidazole or 2-imidazolecarboxaldehyde;
the solvent comprises ultrapure water, methanol, ethanol, N-dimethylformamide.
Preferably, the Zn-MOF nano-delivery vehicle comprises a ZIF-90 nano-delivery vehicle and/or a ZIF-8 nano-delivery vehicle;
the ZIF-90 nano delivery carrier is prepared by mixing and dissolving zinc acetate and 2-imidazole formaldehyde in N, N-dimethylformamide, and self-assembling; the feeding ratio of the zinc acetate, the 2-imidazole formaldehyde and the N, N-dimethylformamide is 1mg: 0.5-10 mg: 0.5-10 mL;
the ZIF-8 nanometer delivery carrier is prepared by dissolving zinc nitrate and 2-methylimidazole in ultrapure water, methanol or ethanol for self-assembly; the feeding ratio of the zinc nitrate, the 2-methylimidazole and water or methanol or ethanol is 1mg: 1-20 mg: 1-10 mL.
Preferably, in the miRNA-loaded Zn-MOF nano-delivery vehicle, the mass ratio of miRNA to Zn-MOF nano-delivery vehicle is (1-2): (7-42).
Preferably, the matrix in the microneedle form is hydrogel formed by crosslinking a photosensitive biological material under the action of a photoinitiator, and is obtained by curing;
the photosensitive biological material comprises at least one of the following: methacrylic acid esterified hyaluronic acid, methacrylic acid anhydrified gelatin and methacryloylated sodium alginate.
Preferably, gold nanoparticles are embedded in the microneedle-shaped matrix;
the mass ratio of the photosensitive biological material to the gold nano particles used in the preparation of the microneedle-shaped matrix is (1-30): 1.
the invention provides an application of the miRNA microneedle patch in preparing a medicament for preventing and/or treating myocardial ischemia reperfusion injury.
The invention provides an application of the miRNA microneedle patch in preparing a medicament for preventing and/or treating arrhythmia caused by myocardial ischemia.
The invention provides a medicament for treating myocardial ischemia reperfusion injury, which comprises a miRNA microneedle patch and medical glue for sticking the miRNA microneedle patch.
The invention provides a miRNA microneedle patch based on a Zn-MOF nano delivery carrier, which comprises a microneedle-shaped matrix and a miRNA-loaded Zn-MOF nano delivery carrier embedded in a needle head part of a microneedle in the matrix; the Zn-MOF nano delivery carrier is combined with miRNA through electrostatic acting force; the miRNA comprises miR-30d, miR-30d mimic or miR-30d agonist. The invention overcomes the problem that miRNA is degraded by various lytic enzymes in lysosomes, takes a Zn-MOF metal organic framework as a gene nano delivery carrier, has acid response characteristic, can be degraded in an acidic environment, and released imidazole molecules can neutralize the acidic environment to deliver loaded cargoes to cytoplasm and present a proton sponge effect. According to the invention, zn-MOF is adopted as a nano delivery carrier of miR-30d, and experiments at a cell level prove that miR-30d can be released from lysosomes and positioned in cytoplasm. The Zn-MOF nano delivery vector-based lysosome escape of miR-30d is realized, and the method has important significance in improving the content of miR-30d in cells. Meanwhile, aiming at the problem that miRNA does not have tissue specificity and is not beneficial to miRNA treatment in specific organs, the miRNA microneedle patch disclosed by the invention is used for delivering miR-30d in a cardiac myocardium without entering a cardiac chamber when being attached to the cardiac myocardium of a heart, so that systemic distribution of miR-30d caused by a cardiac pumping function is avoided, and side effects of miRNA treatment on other normal organs are reduced. Experiments at the cellular level and the animal level prove that miR-30d plays a role in inhibiting myocardial apoptosis and myocardial fibrosis better, and the in-situ delivery of the micro-needle patch to the cardiac muscle of miR-30d improves the curative effect of miR-30d on relieving myocardial ischemia reperfusion injury.
Meanwhile, the Zn-MOF nano delivery carrier in the miRNA microneedle patch provided by the invention has good biocompatibility and can improve the transfection efficiency of miRNA. By analyzing blood biochemical indexes of the serum of the treated mice, it is clear that the Zn-based MOF and the microneedle patch do not influence normal physiological functions of organisms.
Furthermore, the invention also defines that the miRNA microneedle patch is also embedded with nano gold particles. The gold nanoparticles are nontoxic and electrically conductive. After myocardial infarction, although rapid recovery of blood flow can greatly relieve cardiac ischemic injury, in long-term development, negative ventricular remodeling of the left ventricle is still possible, and heart failure is further caused, so that the life of a patient is seriously endangered. The miRNA microneedle patch provided by the invention can be attached to the myocardium of the left ventricle, provides a certain mechanical support, and relieves the ventricular remodeling caused by ischemia from the mechanical support angle. In addition, the miRNA microneedle patch provided by the invention has good conductivity, and can restore the electric signal conduction among myocardial cells, thereby avoiding arrhythmia caused by the interruption of electrocardiosignals. According to the results of the heart failure, the electrocardiogram and the pathology, the heart function of the mice in the treated group is greatly improved and the electrocardiosignal rule is greatly improved compared with the mice in the untreated group after the ischemia reperfusion injury.
In addition, the invention has the following beneficial effects:
(1) MOF nano particles such as ZIF-8 or ZIF-90 are used as gene vectors for myocardial ischemia reperfusion injury treatment for the first time, and compared with viral vectors and liposomes, the gene vectors have lysosome escape capacity, and the curative effect of miRNA in myocardial ischemia reperfusion injury is remarkably improved.
(2) Compared with myocardial injection of a syringe, the microneedle patch can keep an injection site to be a myocardium instead of a heart chamber, realize in-situ continuous administration, and not affect other normal organs. Compared with intravenous injection, the microneedle patch is a limited countermeasure against the multi-target non-specific defect of miRNA, and can reduce toxic and side effects on other normal tissues.
(3) The delivery system prepared by the invention has good biocompatibility, does not influence liver function and kidney function, and does not cause pathological damage to other normal tissues.
(4) Compared with the existing microneedle patch for treating myocardial ischemia reperfusion injury, the invention has the functions of maintaining the mechanical strength of ventricles, has conductivity, can recover the electric conduction signals in injured myocardial tissues, and has important curative effects on improving arrhythmia caused by ischemia.
Drawings
FIG. 1 is an XRD pattern diagram of a ZIF-8 nano delivery system;
FIG. 2 is an XRD pattern for a ZIF-90 nano delivery system;
FIG. 3 is an ultraviolet absorbance spectrum of a miR-30d mimetic;
FIG. 4 is an ultraviolet absorption spectrum of HAMA;
FIG. 5 is an ultraviolet visible absorption spectrum of gold nanoparticles;
FIG. 6 is a graph showing the results of verifying the conductivity of gold nanoparticle-loaded hydrogels;
FIG. 7 shows the cytotoxicity test results of gold nanoparticles;
FIG. 8 is an infrared spectrum of miR-30d@ZIF-8 prepared in example 2;
FIG. 9 is a graph of the particle size distribution of miR-30d@ZIF-8 prepared in example 2;
FIG. 10 is a graph showing the results of the specific surface area of miR-30d@ZIF-8 prepared in example 2;
FIG. 11 is a graph showing the pore size distribution of miR-30d@ZIF-8 prepared in example 2;
FIG. 12 is a TEM image of miR-30d@ZIF-8 prepared in example 2;
FIG. 13 is the cytotoxicity of miR-30d@ZIF-8 prepared in example 2;
FIG. 14 is an SEM image of a HAMA hydrogel microneedle patch prepared according to example 3;
FIG. 15 is an infrared spectrum of a HAMA hydrogel microneedle patch prepared in example 3;
FIG. 16 is a current-potential plot of a HAMA hydrogel microneedle patch prepared in example 3;
FIG. 17 is a graph showing the results of detection of AC16 cell adhesion on the surface of gold nanoparticle-loaded HAMA hydrogel prepared in example 3 using a Calcein/PI dead-time staining kit, and the scale: 100 μm;
FIG. 18 is a graph showing the results of releasing miR-30d@ZIF-8 nanoparticles in PBS pH7.4 from the HAMA hydrogel microneedle patch prepared in example 3;
FIG. 19 is a current-potential diagram of HAMA hydrogel-loaded gold nanoparticles prepared in example 4;
FIG. 20 is a graph showing the growth of AC16 cells adhered to the surface of gold nanoparticle-loaded HAMA hydrogel prepared in example 4 using a Calcein/PI dead-time staining kit, and the scale: 100 μm;
FIG. 21 is an experiment for co-localization of miR-30d@ZIF-8 nanoparticles prepared in example 2 in neonatal rat cardiomyocytes, scale bar: 20 μm;
FIG. 22 shows cardiac fluorescence imaging results of 1 week, 2 weeks, and 3 weeks after the conductive microneedle patch loaded with gold nanoparticles and miR-30d@ZIF-8 nanoparticles prepared in example 3 was transplanted into the heart of a mouse with myocardial ischemia reperfusion injury;
FIG. 23 is a fluorescence microscopy image of heart tissue sections 1 week, 2 weeks, 3 weeks after transplanting the conductive microneedle patch loaded with gold nanoparticles and miR-30d@ZIF-8 nanoparticles prepared in example 3 into the heart of a mouse with myocardial ischemia reperfusion injury;
FIG. 24 shows the Zn element distribution results of tissues such as liver, kidney, spleen, lung, brain, etc. of a mouse after 3 weeks of treatment of myocardial ischemia reperfusion injury with the microneedle patch prepared in example 3;
FIG. 25 is a cardiac hyper-result of improving cardiac function after 3 weeks of treatment of myocardial ischemia reperfusion injury in mice with microneedle patches prepared in example 3 and comparative example 1;
FIG. 26 is a WesternBlot test result of proteins associated with apoptosis of heart tissue of mice treated with the microneedle patch prepared in example 3 and comparative example 1 for 3 days after myocardial ischemia reperfusion injury;
FIG. 27 is a graph showing the effect of the microneedle patch prepared in example 3 and comparative example 1 on liver and kidney function indexes of mice after 3 weeks of treatment of myocardial ischemia reperfusion injury;
FIG. 28 is a graph showing the proportion of myocardial fibrosis calculated from the Marsonian staining of heart Marsonian staining of mice treated with a conductive microneedle patch prepared in example 3 and comparative example 1 and a conductive microneedle patch loaded with miR-30d@ZIF-8 nanoparticles after 3 weeks of myocardial ischemia reperfusion injury;
FIG. 29 is a TUNEL fluorescence staining chart of heart tissue of mice treated with the microneedle patch prepared in example 3 and comparative example 1 after 3 days of myocardial ischemia reperfusion injury;
FIG. 30 is the CX43 immunofluorescence staining results of heart tissue of mice treated with the conductive microneedle patches prepared in example 3 and comparative example 1 and a conductive microneedle patch loaded with a miR-30d@ZIF-8 nano delivery vehicle after 3 weeks of myocardial ischemia reperfusion injury;
fig. 31 is an electrocardiogram of mice 3 weeks after treatment of myocardial ischemia reperfusion injury with the conductive microneedle patches prepared in example 3 and comparative example 1 and a conductive microneedle patch loaded with miR-30d@zif-8 nm delivery vehicle.
Detailed Description
The invention provides a miRNA microneedle patch based on a Zn-MOF nano delivery carrier, which comprises a microneedle-shaped matrix and a miRNA-loaded Zn-MOF nano delivery carrier embedded in a needle head part of a microneedle in the matrix;
the Zn-MOF nano delivery carrier is combined with miRNA through electrostatic acting force;
the miRNA comprises miR-30d, miR-30d mimic or miR-30d agonist.
In the invention, the Zn-MOF nano-delivery carrier is prepared by preferably self-assembling zinc salt and imidazolyl molecules in a solvent. The zinc salt preferably comprises zinc acetate or zinc nitrate. The imidazolyl molecule preferably comprises 2-methylimidazole or 2-imidazolecarboxaldehyde. The solvent comprises ultrapure water, methanol, ethanol, N-dimethylformamide.
In an embodiment of the invention, two Zn-MOF nano-delivery vehicles, namely a ZIF-90 nano-delivery vehicle and a ZIF-8 nano-delivery vehicle, are provided. The ZIF-90 nano delivery carrier is prepared by mixing and dissolving zinc acetate and 2-imidazole formaldehyde in N, N-dimethylformamide, and self-assembling; the charging ratio of the zinc acetate, the 2-imidazole formaldehyde and the N, N-dimethylformamide is preferably 1mg: 0.5-10 mg:0.5 to 10mL, more preferably 1mg: 1-8 mg:0.8 to 8mL, more preferably 1mg: 2-6 mg:1.5 to 6mL, and still more preferably 1mg:4mg:4mL. The temperature of the self-assembly is preferably 20 to 120 ℃, more preferably 50 to 80 ℃, and most preferably 70 ℃. The ZIF-8 nanometer delivery carrier is prepared by dissolving zinc nitrate and 2-methylimidazole in ultrapure water, methanol or ethanol for self-assembly; the charging ratio of the zinc nitrate, the 2-methylimidazole and water or methanol or ethanol is preferably 1mg: 1-20 mg:1 to 10mL, more preferably 1mg: 2-18 mg:1.5 to 9mL, more preferably 1mg: 4-15 mg:2 to 8mL, still more preferably 1mg: 7-12 mg: 4-7 mL, most preferably 1mg:10mg:5mL. The temperature of the self-assembly is preferably 20 to 28 ℃, more preferably 25 ℃. The dissolution is preferably carried out by magnetically stirring for 0.25 to 12 hours, more preferably 0.5 to 10 hours, still more preferably 40 minutes to 8 hours, still more preferably 2 to 6 hours, and most preferably 4 hours.
In the present invention, in the miRNA-loaded Zn-MOF nano-delivery vehicle, the mass ratio of miRNA to Zn-MOF nano-delivery vehicle is preferably (1-2): (7 to 42), more preferably 1: (14-21). The preparation method of the Zn-MOF nano delivery carrier loaded with miRNA is characterized in that the prepared Zn-MOF nano delivery carrier and miR-30d mimic or agonist are preferably respectively dissolved in enzyme-free ultrapure water, mixed according to a proportion, ultrasonically mixed for 1-60min at room temperature, centrifugally washed and freeze-dried. The nucleotide sequence of miR-30d is shown as SEQ ID NO. 1 (UGUAAACAUCCCCGACUGGAAG). In the embodiment of the invention, the nucleotide sequence of the miR-NC serving as a control is shown as SEQ ID NO. 2 (UUUGUACUACACAAAAGUACUG).
In the present invention, the matrix in the form of the microneedle is preferably hydrogel formed by crosslinking a photosensitive biological material under the action of a photoinitiator, and is obtained by curing. The photosensitive biological material comprises at least one of the following: methacrylic acid esterified hyaluronic acid, methacrylic acid anhydrified gelatin and methacryloylated sodium alginate. The photoinitiator is preferably lithiumphenyl (2, 4, 6-trimethylphenyl) phosphinate, and the preparation method of the photoinitiator solution comprises the following steps: 0.025g of the photoinitiator powder was dissolved in 10ml of BS (pH 7.4, 0.01M) in a water bath at 37℃for 30 min. The concentration of the photoinitiator solution was 2.5g/L.
In the present invention, the final concentration of the miRNA-loaded Zn-MOF nano-delivery vehicle in the hydrogel at the time of the microneedle-shaped matrix is preferably (1 to 30): 100, more preferably (10-20): 100, most preferably 14.336:100. In the embodiment of the invention, the concentration of the Zn-MOF nano delivery carrier loaded with miRNA is 14.336mg/mL. The Zn-MOF nano delivery carrier not only has good biocompatibility, but also can improve the transfection efficiency of miRNA. By analyzing blood biochemical indexes of the serum of the treated mice, it is clear that the Zn-based MOF and the microneedle patch do not influence normal physiological functions of organisms.
In the present invention, gold nanoparticles are preferably embedded in the matrix in the form of microneedles. The mass ratio of the photosensitive biological material to the gold nano particles used in the preparation of the microneedle-shaped matrix is (1-40) x 10 4 1, more preferably (1-20). Times.10 4 1, more preferably (5-10). Times.10 4 1, most preferably 8.626X 10 4 :1. In inventive examples 3 and 4, the photoinitiator concentration was 0.01mol/L. The mass fraction of the hydrogel solution was 10%. The concentration of gold nanoparticles was 11.882. Mu.g/mL. The microneedle patch has good conductivity after embedding gold nanoparticles, and can restore the electric signal conduction between myocardial cells, thereby avoiding arrhythmia caused by the interruption of electrocardiosignals. According to the results of the heart failure, the electrocardiogram and the pathology, the heart function of the mice in the treated group is greatly improved and the electrocardiosignal rule is greatly improved compared with the mice in the untreated group after the ischemia reperfusion injury.
In the invention, the preparation method of the miRNA microneedle patch preferably comprises the following steps:
and dissolving a Zn-MOF nano delivery carrier loaded with miRNA in a photoinitiator solution, adding a photosensitive biological material into the system, dissolving, filling the needle point part of a microneedle mould, continuously introducing the photoinitiator solution only dissolved with the photosensitive biological material into the microneedle mould after air is removed, and curing after air is removed to obtain the miRNA microneedle patch.
In the present invention, the height of the tip is preferably 200 μm. The vertical height of the miRNA microneedle patch is preferably 2 mm-1 cm, and more preferably 5mm.
In the present invention, when the gold nanoparticles are further embedded in the microneedle patch, the gold nanoparticles exist at the needle tip, the needle body, and the base, and therefore, the gold nanoparticles are added at the time of two dissolution. In order to ensure that the nano gold particles are uniformly dispersed and in the microneedle patch, the concentration of the nano gold particles is kept consistent during two times of dissolution.
Based on the fact that Zn-MOF in the miRNA microneedle patch has good and efficient delivery effect on active molecule miRNA, and meanwhile, the microneedle structure ensures that the active molecule miRNA plays a role in myocardial layer cells and plays a role in inhibiting myocardial cell apoptosis and myocardial fibrosis, the invention provides application of the miRNA microneedle patch in preparation of medicines for preventing and/or treating myocardial ischemia reperfusion injury.
Based on the high conductivity and non-toxicity of the gold nanoparticles embedded in the miRNA microneedle patch, the invention provides application of the miRNA microneedle patch in preparing medicines for preventing and/or treating arrhythmia caused by myocardial ischemia.
The invention provides a medicament for treating myocardial ischemia reperfusion injury, which comprises a miRNA microneedle patch and medical glue for sticking the miRNA microneedle patch.
In the invention, the miRNA microneedle patch is stuck on the myocardium, and the miRNA microneedle patch utilizes a nano delivery system to efficiently and continuously deliver the active molecule miRNA to the myocardium, thereby being beneficial to reaching focus and playing a role in treating myocardial ischemia reperfusion injury. The medical glue preferably comprises alpha-n-butyl cyanoacrylate or porcine fibrin glue.
According to the application method of the miRNA microneedle patch, blood on a heart is wiped off after a chest opening operation, 0.5 mu L of medical glue is sucked by a pipette and smeared on four corners of one surface of the microneedle patch containing microneedles, and after the microneedles are slightly pressed for 5 seconds, a chest is sutured.
The following describes a miRNA microneedle patch based on Zn-MOF nano-delivery vehicle, and a preparation method and application thereof in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation method of miRNA microneedle patch based on Zn-MOF nano delivery carrier
Preparation method of Zn-MOF nano delivery carrier
Preparation method of ZIF-8 nano delivery carrier
①0.2g(0.66mmol)Zn(NO 3 ) 2 ·6H 2 Adding O into 0.8mL of pure water to be fully dissolved to obtain a solution (1);
(2) 2g of 2-methylimidazole (23.36 mmol) was added to 8mL of pure water and sufficiently dissolved to obtain a solution (2);
(3) dropwise adding the solution (2) into the solution (1), and stirring for 1min at normal temperature;
(4) centrifugal 20 minutes at 10000rpm, washing 3 times with pure water, and freeze drying.
XRD crystal form detection is carried out on the prepared ZIF-8 nanometer delivery carrier, and the result is shown in figure 1. As can be seen from FIG. 1, the crystalline form of ZIF-8 prepared in example 1 was consistent with the simulated and calculated crystalline form of ZIF-8 reported in the literature (ACSAppl. Mater. Interface 2018,10,3,2328-2337), indicating that the examples successfully prepared ZIF-8 nanocarriers.
Preparation method of ZIF-90 nano delivery carrier
①0.1mol/LZn(CH 3 COOH) 2 ·2H 2 Preparing an O solution: 43.9mg Zn (CH) 3 COOH) 2 ·2H 2 O is fully dissolved in 2mLN, N-Dimethylformamide (DMF) to obtain a solution (1);
(2) 0.2 mol/L2-imidazole-formaldehyde solution: 38.4mg of imidazole-2-formaldehyde is fully dissolved in 2mLDMF to obtain solution (2);
(3) solution (1) was added to solution (2), stirred at 500rpm for 5 minutes, 10ml of LDMF was added, and stirred at 70℃for 20 minutes at 500 rpm;
(4) 1000rpm, centrifuging for 20min, discarding supernatant, sequentially washing with DMF for 1 time, washing with absolute ethanol for 2 times, lyophilizing, and storing at 4deg.C.
XRD crystal form detection is carried out on the prepared ZIF-90 nano delivery carrier, and the result is shown in figure 2. As can be seen from FIG. 2, the crystal form of ZIF-90 prepared in example 1 is consistent with the simulated and calculated crystal form of ZIF-90 referred to in the literature (adv. Function. Mater.2022,32,2108603, h in FIG. 1), which shows that the examples successfully prepared ZIF-90 nanocarriers.
2. Construction method of nano delivery carrier based on ZIF-8 and loaded with miR-30d mimic
(1) The miR-30d mimic is synthesized by the Ruibo biological company, and the ultraviolet absorption spectrum is shown in figure 3. Dissolving miR-30d mimic with DEPC water, wherein the concentration is 1 mug/mu L;
(2) the ZIF-8 is fully dissolved by DEPC water ultrasonic treatment for 15min, and the concentration is 1 mug/mu L;
(3) mixing miR-30d mimics and ZIF-8 in a mass ratio of 1:7, 2:7, 1:14, 1:21 and 1:42, carrying out electrostatic adsorption reaction on the mixture at 250rpm for 30 minutes by a shaker at 4 ℃, centrifuging at 10000rpm for 15 minutes, and discarding the supernatant to obtain the product.
3. Preparation method of hydrogel microneedle patch
1. Preparation method of blank hydrogel microneedle
(1) 0.25% photoinitiator solution formulation
0.025g of photoinitiator powder (lithiumphenyl (2, 4, 6-trimethylzolyl)) was dissolved in 10mL of PBS (pH 7.4, 0.01M) in a water bath at 37℃for 30min to give the final product;
(2) weighing 0.1g HAMA, adding 1mL of photoinitiator solution, and dissolving in a water bath at 37 ℃ for 30min to form 10% hydrogel solution;
(3) dropping HAMA hydrogel solution onto microneedle mould, removing bubbles at room temperature under negative pressure, sucking out bubble-containing solution, heating and concentrating in oven at 37deg.C for 5 hr, dropping HAMA hydrogel solution, heating and concentrating at 37deg.C for 5-6 hr, and concentrating with 405nm light source (30 Mw/cm 2 ) Curing for 10s, dripping a small amount of HAMA solution (covering the whole mold surface) three times, heating and concentrating for 12h in a baking oven at 37 ℃, and demoulding to obtain the product.
2. Preparation method of methacryloylated gelatin hydrogel microneedle patch
The microneedle patch was prepared according to the above-described blank hydrogel microneedle preparation method, except that the concentration of methacryloylated gelatin was 5%, i.e., 0.05g gelma plus 1ml fbs was dissolved under the same conditions.
3. Preparation method of miR-30d@ZIF-8 hydrogel microneedle
The microneedle patch was prepared according to the above-described method for preparing a blank hydrogel microneedle, except that a solution containing miR-30d@ZIF-8 was prepared using 0.25% photoinitiator, and the final concentration of miR-30d@ZIF-8 was 14.336mg/mL.
4. Preparation method of miR-30d@ZIF-8 and gold nanoparticle hydrogel microneedle patch
Preparation of gold nanoparticles (AuNP): adding chloroauric acid and sodium citrate into ultrapure water, wherein the feeding ratio of the chloroauric acid to the sodium citrate dihydrate is 50mmol/L and 300mmol/L, performing chemical reduction reaction to obtain gold nanoparticle solution, and performing centrifugal washing and drying to obtain gold nanoparticles. The UV-visible absorption spectrum of the gold nanoparticles is shown in FIG. 5.
Preparing a mixed solution of miR-30d@ZIF-8 and AuNP by using 0.25% of photoinitiator, dissolving HAMA by using the mixed solution to form a hydrogel solution, and preparing the microneedle according to the same conditions. The final concentration of miR-30d@ZIF-8 is 14.336mg/mL. The final concentration of AuNP was 5.941 μg/mL.
The prepared hydrogel microneedle patch is loaded with gold nanoparticles, so that the hydrogel microneedle patch can conduct electricity, a circuit is connected, and a bulb emits light (see figure 6).
To verify the cytotoxicity of gold nanoparticles, after incubation of NRCM (neonatal rat cardiomyocytes) for 24h and 48h with gold nanoparticle solutions of different concentrations, the cell viability was still around 100%, indicating that gold nanoparticles were less cytotoxic to the cardiomyocytes (see fig. 7).
Example 2
miR-30d@ZIF-8 nanoparticle
(1) 0.2g (0.66 mmol) of Zn (NO 3) 2.6H2O was added to 0.8mL of pure water and sufficiently dissolved to obtain a solution (1);
(2) 2g of 2-methylimidazole (23.36 mmol) was added to 8mL of pure water and sufficiently dissolved to obtain a solution (2);
(3) dropwise adding the solution (2) into the solution (1), and stirring for 15 minutes at normal temperature;
(4) centrifuging at 10000rpm for 20min, washing with pure water for 3 times, and freeze drying. Obtaining ZIF-8 nano particles.
(5) Dissolving the miR-30d mimic with DEPC water to obtain a final concentration of 1 mug/mu L;
(6) the ZIF-8 is fully dissolved by DEPC water in an ultrasonic mode for 15min, and the final concentration is 1 mug/mu L;
(7) mixing miR-30d simulative substances with a corresponding volume with ZIF-8 according to the mass ratio of miR-30d simulative substances to ZIF-8 of 1:21, reacting for 30min at 250rpm on a shaking table at 4 ℃, and centrifuging at 10000rpm multiplied by 15min to remove supernatant.
And carrying out morphological characterization by respectively carrying out infrared spectrum, dynamic light scattering analysis, BET specific surface area analysis and aperture analysis and transmission electron microscope imaging, and carrying out MTT cell viability experiments to detect toxicity of the ZIF-8 nanometer delivery carrier.
The results are shown in fig. 8 to 13: miR-30d mimics have been successfully loaded into ZIF-8 nano delivery vehicles, and have particle sizes between 100-200nm, and exhibit a porous structure. Through MTT cytotoxicity experiments, it is clear that the ZIF-8 nano delivery vector does not significantly inhibit the survival rate of AC16 cells, indicating that ZIF-8 has lower myocardial cytotoxicity.
Example 3
Preparation method of conductive microneedle patch loaded with gold nanoparticles and miR-30d@ZIF-8 nanoparticles
Dissolving miR-30d@ZIF-8 nano particles (with the concentration of 14.336 mg/mL) and gold nano particle solution (with the concentration of 11.882 mu g/mL) by using 0.25% photoinitiator solution, dissolving 0.1g of HAMA in 1mL of solution to form a hydrogel solution, and preparing the microneedle patch according to the same conditions of the embodiment 1.
And carrying out appearance characterization analysis on the microneedle patch through scanning electron microscope imaging, infrared spectrum and electrochemical analysis. Fig. 14 to 16 show the microstructure characterization results of the microneedle patch, respectively. The chemical structure of the HAMA microneedle patch is determined through a scanning electron microscope and an infrared spectrum, and the CV curve of the conductive microneedle patch is analyzed by adopting an electrochemical workstation, so that the hydrogel material loaded with gold nanoparticles has certain conductivity.
To further determine the toxic side effects on cardiomyocytes when the microneedle patch was adhered to the myocardium, three-dimensional culture of AC16 cells was performed on the hydrogel surface, staining of AC16 cells by Calcein/PI dead-alive staining kit, green fluorescence showed living cells, red fluorescence showed dead cells, and the ratio of red-green fluorescence was observed, demonstrating that a large number of AC16 cells survived on the hydrogel surface, thus showing that AC16 cells were able to adhere to the HAMA hydrogel surface and continued to grow (see fig. 17).
In order to evaluate the release of miR-30d@ZIF-8 nanoparticles from the hydrogel, a hydrogel kinetic experiment was performed. FIG. 18 is a kinetic profile of in vitro simulated miR-30d@ZIF-8 nanoparticles released in HAMA hydrogel. The results showed that miR-30d@ZIF-8 nanoparticles were released slowly in PBS pH7.4 and completely on day 16.
Example 4
Preparation method of conductive microneedle patch loaded with gold nanoparticles and miR-NC@ZIF-8 nanoparticles
Dissolving miR-NC@ZIF-8 nano particles (the concentration is 14.336mg/mL, miR-NC mimics are nonsensical miRNA in disorder and serve as negative control) and gold nano particle solution (11.882 mug/mL) by using 0.25% photoinitiator solution, dissolving 0.1g of HAMA in 1mL of solution to form hydrogel solution, and preparing the micro needle according to the same conditions.
Fig. 19 shows the CV curve of the conductive microneedle patch analyzed using an electrochemical workstation, and compared with fig. 16, it was confirmed that the higher the gold nanoparticle concentration, the more conductive the hydrogel material.
To further determine the effect on cell growth when microneedle patches were adhered to cardiomyocytes, AC16 cells were stained with the Calcein/PI dead-alive staining kit, green fluorescence showed living cells, red fluorescence showed dead cells, and AC16 cells three-dimensionally cultured on the hydrogel surface were found to survive in large amounts by the ratio of red-green fluorescence, confirming that AC16 cells were able to adhere to the HAMA hydrogel surface and continued to grow (fig. 20).
Comparative example 1
Preparation method of gold nanoparticle-loaded conductive microneedle
Dissolving gold nanoparticle solution (with the concentration of 5.941 mu g/mL) by using 0.25% photoinitiator solution, dissolving 0.1g of HAMA by using 1mL of solution to form hydrogel solution, and preparing the micro needle according to the same conditions.
Example 5
Experiment of co-localization of miR-30d@ZIF-8 nanodelivery vehicle prepared in example 2 in neonatal rat cardiomyocytes
The new-born rat myocardial cells (NRCM) were extracted and cultured in a gelatin-plated petri dish, 8000 cells per well, and after culturing the cells in a myocardial cell culture medium for 12 hours, miR-30d-Cy3@ZIF-8 nanoparticles (20. Mu.g/mL) prepared in example 1 were added thereto, and after incubation for 4 hours and 24 hours, respectively, hoechst dye and lysosome dye were added to incubate the cells for 10 minutes, and the cells were washed 3 times with PBS and observed under a laser confocal microscope.
The results in fig. 21 show that at 4h incubation, the red fluorescence in NRCM mostly coincides with the green fluorescence, showing a yellow fluorescence, wherein the red fluorescence is from Cy 3-labeled miR-30d-cy3@zif-8 nanoparticles, the green fluorescence indicates lysosomes, and the blue indicates nuclei. When incubated for 24 hours, the red fluorescence and the green fluorescence in NRCM dispersed at different positions of the cells, indicating that miR-30d-Cy3@ZIF-8 nanoparticles had escaped from lysosomes. This result demonstrates that the ZIF-8 nano delivery vehicle can help miR-30d escape from lysosomal degradation, thereby improving the efficacy of miR-30 d.
Example 6
The conductive microneedle patch loaded with gold nanoparticles and miR-30d@ZIF-8 nanoparticles prepared in example 3 was subjected to in-vivo fluorescence imaging after implantation in the heart of a mouse
The myocardial ischemia reperfusion injury model is constructed by using 8 weeks of C57 male mice, meanwhile, the conductive microneedle patch loaded with gold nanoparticles and miR-30d@ZIF-8 nanoparticles prepared in the example 2 is transplanted to the heart of the mice, and after 1 week, 2 weeks and 3 weeks of treatment, the heart of the mice is taken to be placed under a living body fluorescence imager for observing fluorescence distribution.
The results are shown in FIG. 22. The fluorescent signal in the heart of the mice persisted for 3 weeks, but the signal intensity gradually decreased with increasing time, indicating that the miR-30d@ZIF-8 nanoparticles were metabolized out of the body during treatment.
The staining of heart tissue sections collected for 3 weeks is observed under a fluorescence microscope, and as shown in fig. 23, the red fluorescence signal of miR-30d@ZIF-8 and the green fluorescence signal of gold nanoparticles are both found to coincide with the positions of myocardial cells, which indicates that the microneedle patch is favorable for the local sustained release of miR-30d@ZIF-8 nanoparticles and gold nanoparticles in myocardial cells of the left ventricle wall.
Example 7
Experiment for improving myocardial ischemia reperfusion injury of mice by using gold nanoparticle-and miR-30d@ZIF-8 nanoparticle-loaded HAMA hydrogel microneedle patch prepared in example 3 and gold nanoparticle-and miR-NC@ZIF-8-loaded HAMA hydrogel microneedle patch prepared in example 4
Mice myocardial ischemia reperfusion injury model was constructed using 8 week-old C57 male mice, and the successfully modeled mice were randomly divided into 3 groups: one group was a negative control group (the mouse myocardial ischemia reperfusion injury model was not treated), one group was a conductive type microneedle carrying gold nanoparticles and miR-NC@ZIF-8 prepared in example 4 for treating the mouse myocardial ischemia reperfusion injury model (conductive type microneedle group), and one group was a conductive type microneedle carrying gold nanoparticles and miR-30d@ZIF-8 nanoparticles prepared in example 3 for treating the mouse myocardial ischemia reperfusion injury model (conductive type microneedle+miR-30 d@ZIF-8 group). Another group of healthy mice was a positive control group. The heart tissue of the mice is taken at 3 days of treatment to detect myocardial apoptosis, and the heart function of the mice is detected after 3 weeks of treatment.
And (3) carrying out aqua regia digestion, ammonia water neutralization and ultrapure water volume fixing on tissues such as liver, kidney, spleen, lung and brain of a mouse after 3 weeks of treatment by using the microneedle patch, and detecting the content of Zn element in each sample by adopting ICP-OES (inductively coupled plasma-optical emission system), wherein the distribution of Zn element represents the distribution of ZIF-8 as Zn is a main component element of ZIF-8. As shown in fig. 24, the concentration of Zn element in the liver, kidney, spleen, lung and brain of each experimental group of mice is not greatly different, which indicates that miR-30d@zif-8 in the conductive microneedle patch is not dispersed to normal tissues such as liver, kidney, spleen, lung and brain, and also proves the local targeted delivery effect of the microneedle patch on miR-30 d.
The mice myocardial ischemia reperfusion model was treated with miR-30d@ZIF-8 nanoparticle-loaded conductive microneedles and conductive microneedle patches prepared in example 3 and comparative example 1, and after 3 weeks of treatment, the heart function of the mice was detected by using a heart super-detector. As shown in fig. 25, by comparing LVEF and LVFS of each group of mice, it was confirmed that the conductive microneedle loaded with miR-30d@zif-8 nanoparticles can significantly improve cardiac function of mice (significant improvement of LVEF and LVFS), and has a good therapeutic effect.
The conductive microneedle patches prepared in example 3 and comparative example 1 and the conductive microneedles loaded with miR-30d@zif-8 nanoparticles were used to treat a mouse myocardial ischemia reperfusion model for 3 days, and expression of apoptosis-related proteins in heart tissue was analyzed using the WesternBlot method. According to the expression results of the apoptosis-related proteins, the ratio of clear-caspase 3/GAPDH to Bax/GAPDH is compared, and the result is shown in figure 26, which proves that the conductive microneedle loaded with miR-30d@ZIF-8 nanoparticles can obviously inhibit myocardial apoptosis.
Serum from mice treated for 3 weeks was used for detection of liver and kidney function indexes. As shown in fig. 27, the conductive microneedles prepared in example 2 and example 4 did not cause acute injury to the liver and kidney of mice by comparing the positive control group with the negative control group, which indicates that the microneedle patch and ZIF-8 nano-carrier prepared in the invention have good biocompatibility and do not cause toxic or side effects on normal organs.
Comparative example 2
Applicant has revealed in earlier research results that miR-30d is able to inhibit myocardial apoptosis and fibrosis following myocardial infarction (Circulation Research,2021,128, e1-e23; eBioMedicine,2022,81,104108). The miR-30d@ZIF-8 nanoparticle loaded by the conductive microneedle patch is constructed, so that the miR-30d myocardial local delivery is realized, and the effect of inhibiting myocardial fibrosis is achieved. Compared with the conductive micro-needle patch loaded with miR-30d@ZIF-8 nano particles, the conductive micro-needle patch has a lower effect on inhibiting myocardial cell apoptosis and myocardial fibrosis than the conductive micro-needle patch loaded with miR-30d@ZIF-8 nano particles.
Control experiment 1
Constructing a myocardial ischemia reperfusion injury model of a mouse, respectively adhering a conductive micro-needle patch loaded with miR-NC@ZIF-8 and a conductive micro-needle patch loaded with miR-30d@ZIF-8 nano particles to the left ventricle wall of the mouse, and taking the heart of the mouse for histological detection after 3 weeks of treatment.
As shown in fig. 28, the fibrosis ratio of the left ventricle of the mouse was analyzed by masson staining and sirius red staining, and it was found that the conductive microneedle patch carrying miR-30d@zif-8 nanoparticles had a lower myocardial fibrosis ratio (8.03±3.6%) after being adhered to the left ventricle wall of the mouse, whereas the conductive microneedle patch carrying miR-nc@zif-8 had a myocardial fibrosis ratio of 10.32±3.9% after being adhered to the left ventricle wall of the mouse.
As shown in fig. 29, after 3 days of treatment, the proportion of myocardial apoptosis after the conductive microneedle patch carrying miR-30d@zif-8 nanoparticles was adhered to the left ventricle wall of the mouse was 7.58±2.09%, and the proportion of myocardial apoptosis after the conductive microneedle patch carrying miR-nc@zif-8 was adhered to the left ventricle wall of the mouse was 15.32±2.91%, which indicates that miR-30d can significantly reduce myocardial apoptosis and myocardial fibrosis after myocardial ischemia reperfusion.
(2) After myocardial infarction, the electrical conduction signals between myocardial cells are cut off, and arrhythmia is easily induced. The conductive microneedle patch contains gold nano particles, so that the electrophysiological signals of myocardial cells can be recovered, and arrhythmia after myocardial infarction can be improved.
Control experiment 2
Constructing a myocardial ischemia reperfusion injury model of a mouse, respectively adhering a conductive micro-needle patch loaded with miR-NC@ZIF-8 and a conductive micro-needle patch loaded with miR-30d@ZIF-8 nano particles to the left ventricle wall of the mouse, treating for 3 weeks, preparing an electrocardiogram of the mouse, and taking the heart of the mouse to carry out immunofluorescence staining of gap junction protein CX43 on heart tissues.
As a result, as shown in FIG. 30, the expression level of the mouse CX43 protein in the negative control group was (5.12.+ -. 3.4). Times.10 5 miR-NC@ZIF-8 loaded conductive microneedle patch-treated mice have CX43 protein expression level of (19.13+/-12.12) x 10 5 The expression level of the mouse CX43 protein treated by the conductive microneedle patch loaded with miR-30d@ZIF-8 nano particles is (23.3+/-4.02) multiplied by 10 5 . High expression of CX43 protein indicates enhanced electrical conductivity between cardiomyocytes.
Results as shown in fig. 31, the electrocardiogram of mice of the negative control group showed significant arrhythmia, while the electrocardiogram of mice of the treatment group showed regular heart rhythm. The invention determines that the conductive microneedle patch can promote CX43 protein expression and can improve arrhythmia through electrocardiogram.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A miRNA microneedle patch based on a Zn-MOF nano-delivery vehicle, characterized by comprising a matrix in the form of a microneedle and a miRNA-loaded Zn-MOF nano-delivery vehicle embedded in the needle portion of the microneedle in the matrix;
the Zn-MOF nano delivery carrier is combined with miRNA through electrostatic acting force;
the miRNA comprises miR-30d, miR-30d mimic or miR-30d agonist.
2. The miRNA microneedle patch of claim 1, wherein the Zn-MOF nanodelivery vehicle is prepared by self-assembling zinc salts and imidazolyl molecules in a solvent.
3. The miRNA microneedle patch of claim 1, wherein the zinc salt comprises zinc acetate or zinc nitrate;
the imidazolyl molecule comprises 2-methylimidazole or 2-imidazolecarboxaldehyde;
the solvent comprises ultrapure water, methanol, ethanol, N-dimethylformamide.
4. The miRNA microneedle patch of claim 1, wherein the Zn-MOF nanodelivery vehicle comprises a ZIF-90 nanodelivery vehicle and/or a ZIF-8 nanodelivery vehicle;
the ZIF-90 nano delivery carrier is prepared by mixing and dissolving zinc acetate and 2-imidazole formaldehyde in N, N-dimethylformamide, and self-assembling; the feeding ratio of the zinc acetate, the 2-imidazole formaldehyde and the N, N-dimethylformamide is 1mg: 0.5-10 mg: 0.5-10 mL;
the ZIF-8 nanometer delivery carrier is prepared by dissolving zinc nitrate and 2-methylimidazole in ultrapure water, methanol or ethanol for self-assembly; the feeding ratio of the zinc nitrate, the 2-methylimidazole and water or methanol or ethanol is 1mg: 1-20 mg: 1-10 mL.
5. The miRNA microneedle patch according to claim 1, wherein in the miRNA-loaded Zn-MOF nano-delivery vehicle, the mass ratio of miRNA to Zn-MOF nano-delivery vehicle is 1: (1-2): (7-42).
6. The miRNA microneedle patch according to claim 1, wherein the matrix in the microneedle form is hydrogel formed by crosslinking a photosensitive biological material under the action of a photoinitiator, and is obtained by curing;
the photosensitive biological material comprises at least one of the following: methacrylic acid esterified hyaluronic acid, methacrylic acid anhydrified gelatin and methacryloylated sodium alginate.
7. The miRNA microneedle patch of any one of claims 1-6, wherein gold nanoparticles are embedded in the microneedle shaped matrix;
the mass ratio of the photosensitive biological material to the gold nano particles used in the preparation of the microneedle-shaped matrix is (1-30): 1.
8. use of the miRNA microneedle patch of any one of claims 1-7 for the preparation of a medicament for preventing and/or treating myocardial ischemia reperfusion injury.
9. The use of the miRNA microneedle patch of claim 7 in the preparation of a medicament for preventing and/or treating arrhythmia caused by myocardial ischemia.
10. A medicament for treating myocardial ischemia reperfusion injury, which is characterized by comprising the miRNA microneedle patch according to any one of claims 1-7 and medical glue for pasting the miRNA microneedle patch.
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