CN116270706B - ZIF-8-CeO2-MiR200c nano-enzyme - Google Patents
ZIF-8-CeO2-MiR200c nano-enzyme Download PDFInfo
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- CN116270706B CN116270706B CN202310065370.2A CN202310065370A CN116270706B CN 116270706 B CN116270706 B CN 116270706B CN 202310065370 A CN202310065370 A CN 202310065370A CN 116270706 B CN116270706 B CN 116270706B
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- 108091074450 miR-200c stem-loop Proteins 0.000 claims abstract description 28
- ONDPHDOFVYQSGI-UHFFFAOYSA-N zinc nitrate Chemical compound [Zn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ONDPHDOFVYQSGI-UHFFFAOYSA-N 0.000 claims abstract description 26
- LXBGSDVWAMZHDD-UHFFFAOYSA-N 2-methyl-1h-imidazole Chemical compound CC1=NC=CN1 LXBGSDVWAMZHDD-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000002105 nanoparticle Substances 0.000 claims abstract description 9
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- 239000002244 precipitate Substances 0.000 claims description 12
- HSJPMRKMPBAUAU-UHFFFAOYSA-N cerium(3+);trinitrate Chemical compound [Ce+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O HSJPMRKMPBAUAU-UHFFFAOYSA-N 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 8
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
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- BMMGVYCKOGBVEV-UHFFFAOYSA-N oxo(oxoceriooxy)cerium Chemical compound [Ce]=O.O=[Ce]=O BMMGVYCKOGBVEV-UHFFFAOYSA-N 0.000 description 1
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/244—Lanthanides; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/16—Emollients or protectives, e.g. against radiation
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
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- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Dermatology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Toxicology (AREA)
- Inorganic Chemistry (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses ZIF-8-CeO 2 -miR200c nano-enzyme, and a preparation method and application thereof. The invention provides a preparation method of a nano enzyme containing miR200C, which comprises the steps of firstly preparing CeO 2 nano particles, then adding miR200C, a 2-methylimidazole solution and a zinc nitrate solution in sequence, carrying out sequential self-assembly to obtain a compound, and then encapsulating the compound by using C18PMH-PEG to synthesize ZIF-8-CeO 2 -miR200C. The ZIF-8-CeO2-miR200c nano-enzyme prepared by the invention can promote proliferation, migration, angiogenesis and apoptosis resistance of HaCaT and HUVEC cells, so that the in-vivo and in-vitro synergistic radiation resistance is exerted. Provides an important reference for the biological material treatment of RISI.
Description
Technical Field
The invention relates to the technical field of RISI (RISI) control, in particular to ZIF-8-CeO 2 -miR200c nano-enzyme, and a preparation method and application thereof.
Background
Radioactive skin injury (RISI) is a common complication of nuclear Radiation and clinical radiotherapy, and no specific treatment method exists clinically at present. However, skin radiation reactions are common side effects that limit the dose and therapeutic effect of radiation therapy. In cancer patients receiving radiation therapy, skin reactions occur up to 95% and serious skin lesions occur in nearly 10%. Currently, the exact mechanism of RISI is not cleared and standardized methods for effective prevention and treatment are lacking.
The CeO 2 NPs nano particles are relatively stable, and have the characteristics of good biocompatibility, low toxicity, low cost, environmental friendliness and the like. Ce has two main oxidation states, including tetravalent (Ce 4+) and trivalent (Ce 3+）,CeO2 NPs have cubic fluorite structures, and surface symbiotic Ce 3+ and Ce 4+. Cerium oxide (CeO 2) has the main advantage of generating oxygen (O) vacancies in the structure, thus, it helps treat various oxidative stress related diseases, as a metal organic framework, ZIF-8 consists of zinc ions linked to N atoms in 2-methylimidazole, ZIF-8 nanoparticles are multifunctional nanocarriers, regulating functional nucleic acids such as DNA zyme, ZIF-8 nanoparticles by electrostatic and ligand interactions can achieve tumor targeted accumulation of nucleic acid payloads by enhancing permeability and retention effects, and promoting uptake of nucleic acids by cells without degradation.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide ZIF-8-CeO 2 -miR200c nano-enzyme, and a preparation method and application thereof. The ZIF-8-CeO2-miR200c nano-enzyme prepared by the invention can promote proliferation, migration, angiogenesis and apoptosis resistance of HaCaT and HUVEC cells, so that the in-vivo and in-vitro synergistic radiation resistance is exerted. Provides an important reference for the biological material treatment of RISI.
In order to achieve the above purpose, the invention adopts the following technical scheme:
In a first aspect of the invention, there is provided the use of miR200c for preparing a control RISI product.
In a second aspect of the invention, there is provided a ZIF-8-CeO 2 -miR200c nanoenzyme prepared by the following method:
(1) Dispersing CeO 2 nano particles in deionized water to obtain CeO 2 dispersion, dissolving miR200c in the deionized water to obtain miR200c solution, adding miR200c solution into CeO 2 dispersion, stirring, then adding 2-methylimidazole solution, continuously stirring, then adding zinc nitrate solution for reaction, centrifuging and washing precipitate after the reaction is finished;
(2) Dissolving C18PMH-PEG in ultrapure water, adding the precipitate obtained in the step (1), performing ultrasonic treatment in ice bath, and centrifuging to obtain the ZIF-8-CeO 2 -miR200C nano enzyme.
Preferably, in the step (1), the concentration of the CeO 2 dispersion liquid is 10mg/mL, and the concentration of the miR200c solution is 20OD/mL.
Preferably, in the step (1), the concentration of the 2-methylimidazole solution is 20mg/ml, and the concentration of the zinc nitrate solution is 100mg/ml.
Preferably, in the step (1), the volume ratio of the CeO 2 dispersion liquid, the miR200c solution, the 2-methylimidazole solution and the zinc nitrate solution is 1:1:1:2.
Preferably, in step (1), the reaction time is 30min.
Preferably, in the step (1), the CeO 2 nano-particles are prepared by mixing cerium nitrate solution and polyallylamine hydrochloride at room temperature, and centrifugally washing to obtain CeO 2 NP.
The concentration of the cerium nitrate solution was 3.5mg/mL, and the concentration of the polyallylamine hydrochloride was 37.4mg/mL.
MiR200c is purchased from Shanghai Ji Ma pharmaceutical technologies Co.
Forward strand AACAUUCAACGCUGUCGGUGAGU (shown as SEQ ID NO. 1) and reverse strand UCACCGACAGCGUUGAAUGUUU (shown as SEQ ID NO. 2).
Note that "T" represents uracil in the RNA sequence and thymine in the DNA sequence, as specified by the WIPO ST.26 standard. Uracil U represented by 'T' in sequence 1 and sequence 2 in a sequence table of the invention.
Preferably, in the step (2), the addition amount ratio of the C18PMH-PEG, the ultrapure water and the precipitate is 100 mg/5 mL/10 mg.
Preferably, in the step (2), the time of the ultrasonic treatment is 90min, the power is 1000w, and the centrifugation is 21000g for 3h.
In a third aspect of the invention, there is provided an application of ZIF-8-CeO 2 -miR200c nanoenzyme in at least one of the following 1) to 2):
1) Preparing a medicament for preventing and treating RISI;
2) Preparing RISI wound repairing product.
The invention has the beneficial effects that:
(1) According to the invention, the zeolite imidazole ester framework material is used as a carrier, ZIF-8-CeO2-miR200C nano enzyme is synthesized through sequential self-assembly, and then the composite is encapsulated by C18PMH-PEG, so that ZIF-8-CeO2-miR200C is synthesized, the dispersibility and stability of the composite are improved, and miRNA is protected from RNase degradation.
(2) The ZIF-8-CeO2-miR200c nano-enzyme prepared by the invention can promote proliferation, migration, angiogenesis and apoptosis resistance of HaCaT and HUVEC cells, so that the in-vivo and in-vitro synergistic radiation resistance is exerted. Provides an important reference for the biological material treatment of RISI.
Drawings
FIG. 1 is a scanning electron microscope image of (A) ZIF-8-CeO 2 -miR200c and (B) ZIF-8-CeO 2 -miR200 c;
FIG. 2 shows particle size and potential analysis of ZIF-8-CeO 2 -miR200C, (A) Zeta point distribution diagram of ZIF-8-CeO 2 -miR200C, (B) Zeta point distribution diagram and specific information of ZIF-8-CeO 2 -miR200C, (C) particle size of ZIF-8-CeO 2 -miR200C, and (D) particle size and specific information of ZIF-8-CeO 2 -miR 200C;
FIG. 3 biocompatibility and antibacterial activity of ZIF-8-CeO 2 -miR200C, (A) FITC/DAPI image of ZIF-8-CeO 2 -miR200C co-cultured with HaCaT cells, (B) CCK-8 assay of ZIF-8-CeO 2 -miR200C co-cultured with HaCaT cells, (C) G+ (MRSA) and G- (E.coli) bacterial antibacterial activity, (D) bacterial count in C. P <0.05;
FIG. 4 in vitro cytological studies of ZIF-8-CeO 2 -miR200C, (A) in vitro tube formation assays, (B) number of blood vessels in A, (C) effect of material on HaCaT cell migration, (D) percentage of cell mobility in C, < p 0.05;
FIG. 5 ZIF-8-CeO 2 -miR200C was in RISI mouse model, A) wound images on days 1 and 14, (B) wound area on day 14, (C) total recovery time.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background section, conventional drug delivery systems have limited application because of the occurrence of localized side effects such as burning, skin irritation, greasiness, stinging, pruritic rashes, erythema and tenderness. Liposomes, NPs and hydrogels require special storage conditions and phospholipids are less stable in liposome preparation due to redox reactions. There is a need for a safe, efficient RISI repair material.
Based on the above, the invention aims to provide ZIF-8-CeO 2 -miR200c nano-enzyme, and a preparation method and application thereof. The CeO 2 nano enzyme is immobilized on the surface of the ZIF-8 to obtain good catalase activity and prevent aggregation. ZIF-8-CeO 2 exhibited significant ROS resistance in physiological pH or weakly acidic wound microenvironment. In addition, both ZIF-8 and miR200c are negatively charged, so the presence of positively charged CeO 2 reduces their electrostatic repulsion. miR200c is loaded by ZIF-8-CeO 2, as miR200c can reduce DNA damage-induced apoptosis by directly targeting RAD17 and modulating the CHK2 pathway. And then encapsulating the complex by using C18PMH-PEG to synthesize ZIF-8-CeO 2 -miR200C so as to improve the dispersibility and stability of the complex and protect miRNA from degradation.
The nano-carrier with the ZIF-8 core-shell structure is suitable for drug delivery through wound surfaces, and miR200c generated by the nano-carrier is embedded into nano-transfer bodies to promote effective permeation and delivery into stratum corneum wound surfaces. The problems of the prior miR200c are effectively solved, the problems include poor systemic absorption, low bioavailability and high systemic clearance after oral administration. Experimental results show that ZIF-8-CeO 2 -miR200c has good free radical scavenging capability and insignificant cytotoxicity, effectively promotes cell migration, vascularization, resists apoptosis, reduces intracellular ROS, and shows that the material can be effectively applied to protecting RISI.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present application, the technical scheme of the present application will be described in detail with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and are commercially available.
The C18PMH-PEG is purchased from Kangzhou Fisher Biotechnology Co., ltd, and can be prepared by referring to a preparation method of a C18-PMH-PEG compound disclosed in patent CN 110408041B;
miR200c is purchased from Shanghai Ji Ma pharmaceutical technologies, inc., unit 20OD.
Examples
(1) Synthesis of CeO 2 NP
Cerium nitrate solution (3.5 mg/mL) and polyallylamine hydrochloride solution (37.4 mg/mL) were mixed at room temperature in a volume ratio of 9:1 to react, and the centrifuged solid was washed with double distilled water to give CeO 2 NP.
(2) Synthesis of ZIF-8-CeO 2 -miR200c
And (3) dispersing the CeO 2 NP obtained in the step (1) in deionized water to obtain 10mg/ml CeO 2 dispersion. miR200c of 20OD was dissolved in 1mL of water to give miR200c solution. And adding the miR200c solution into the CeO2 dispersion liquid, and stirring for 3 hours. Then 20mg/ml of 2-methylimidazole solution was added, stirred for 30 minutes, and then 100mg/ml of zinc nitrate solution was added. The volume ratio of CeO 2 dispersion liquid, miR200c solution, 2-methylimidazole solution and zinc nitrate solution is 1:1:1:2. After 30 minutes of reaction, the precipitate was centrifuged and washed, dispersed in water and stored in the dark at 4 ℃. 10mg precipitate and 100mg of C18PMH-PEG were added to 5mL of ultrapure water. After ultrasonic treatment in an ice bath for 90 minutes, the unstable nanocomposite was removed by centrifugation at 21000 Xg for 3 hours to obtain ZIF-8-CeO 2 -miR200c nanoenzyme.
The transmission electron microscope was used to observe the morphology of ZIF-8-CeO 2 -miR 129. Particle size and potential were assessed using dynamic light scattering. Fourier transform infrared spectra were recorded using a Nicolet 6700 FTIR spectrometer (4000-600 cm). The elemental composition of the material was analyzed by Energy Dispersive Spectroscopy (EDS)/X-ray photoelectron spectroscopy (XPS). FIG. 1A shows TEM imaging characteristics of ZIF-8-CeO 2 -miR200c after encapsulation with a ZIF-8 shell. Fourier transform infrared (FT-IR) spectra showed that the typical absorption peaks for CeO 2, ZIF-8 and miRNA were shown in the ZIF-8-CeO 2 -miR200c particles (FIG. 1B). The average particle size of ZIF-8-CeO 2 was 1294nm and the zeta potential was 34.6mV (FIG. 2), and these characterizations indicated that the material had been successfully synthesized.
ZIF-8 maintains colloidal stability as compared to the uncoated CeO 2 NPs. TEM and SEM images showed a clean and smooth surface of ZIF-8, although discrete black spots on the surface of ZIF-8 were clearly visible in the presence of CeO 2, high resolution TEM also indicated that drug-loaded ZIF-8 successfully immobilized the CeO 2 nanoparticles to the drug carrier surface.
Comparative example 1
(1) Synthesis of CeO 2 NP
Cerium nitrate solution (3.5 mg/mL) and polyallylamine hydrochloride solution (37.4 mg/mL) were mixed at room temperature in a volume ratio of 9:1 to react, and the centrifuged solid was washed with double distilled water to give CeO 2 NP.
(2) Synthesis of ZIF-8-CeO 2
And (3) dispersing the CeO 2 NP obtained in the step (1) in deionized water to obtain 10mg/ml CeO 2 dispersion. 20mg/ml of 2-methylimidazole solution was added, stirred for 30 minutes, and then 100mg/ml of zinc nitrate solution was added, with the volume ratio of CeO 2 dispersion, 2-methylimidazole solution and zinc nitrate solution being 1:1:2. After 30 minutes of reaction, the precipitate was centrifuged and washed, dispersed in water and stored in the dark at 4 ℃.10 mg precipitate and 100mg of C18PMH-PEG were added to 5mL of ultrapure water. After sonication in an ice bath for 90 minutes, the unstable nanocomposite was removed by centrifugation at 21000 Xg for 3 hours to give ZIF-8-CeO 2.
Comparative example 2
Synthesis of ZIF-8miR200c
Dissolving miR200c with the concentration of 20OD in 1mL of water to obtain miR200c solution, adding 20mg/mL of 2-methylimidazole solution, stirring for 30 minutes, and then adding 100mg/mL of zinc nitrate solution, wherein the volume ratio of miR200c solution to 2-methylimidazole solution to zinc nitrate solution is 1:1:2. After 30 minutes of reaction, the precipitate was centrifuged and washed, dispersed in water and stored in the dark at4 ℃. 10 mg precipitate and 100mg of C18PMH-PEG were added to 5mL of ultrapure water. After sonication in an ice bath for 90 minutes, the unstable nanocomposite was removed by centrifugation at 21000 Xg for 3 hours to give ZIF-8-miR200c.
Test example 1 in vitro cytological test
1. Test procedure
The biological compatibility evaluation and the cell morphology observation are that the cytology experiment is divided into 5 groups, wherein the A group is a normal cell group, the B group is a normal cell+5Gy irradiation group, the C group is a normal cell+5Gy irradiation+ZIF-8-CeO 2-miR200C group, the D group is a normal cell+5Gy irradiation+ZIF 8-miR200C group, and the E group is a normal cell+5Gy irradiation+ZIF 8-CeO 2 group. Cytotoxicity was assessed using the CCK-8 assay. HaCat cells were seeded at a density of 2×10 3 cells/well into group a-C in DMEM supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin at 37 ℃.
After 1,4 and 7 days incubation, cell viability was quantified using the CCK-8 assay. To investigate the effect of cytoskeletal morphology, haCat cells were seeded on group a-E medium for 24 hours, and then the cells were washed 3 times. Washed with pre-warmed phosphate buffered saline and fixed with pre-warmed 4% paraformaldehyde at room temperature. The fixed cells were washed with phosphate buffer and soaked. The cells were then incubated with phalloidin at 37 ℃ for 30 min and 5 min without light and DAPI (2- (4-amidinophenyl) -6-indolecarboxamide dihydrochloride). Fluorescence image scanning microscopy (780, zeiss, germany) of stained cells was obtained using confocal lasers. Fluorescent observations were then performed by staining HaCat cells with DAPI and phalloidin.
Scratch migration HaCaT was grown to confluency in complete medium and seeded in 24 well plates (2X 10 4/well). The 24 hour examination was performed with a pipette, tip and Zeiss video microscope through a monolayer (0 h). The individual experiments were repeated five times per group and specific measurements were performed using ImageJ 1.48v software (NIH, USA). The assay was repeated three times.
In vitro tube formation assay mixed 2X 10 4 HaCaT cells were seeded onto matrigel coated 96-well plates and then added to groups A-E. After 24 hours of stimulation, the newly formed tube was photographed with an inverted microscope (Olympus, USA). Each group was repeated five times.
Bacteria co-culture MRSA (methicillin-resistant Staphylococcus aureus) and Escherichia coli (Escherichia coli) were purchased from the university of army medical science. Bacteria were amplified (grown overnight) to 1X 10 9 Colony Forming Units (CFU)/mL and then diluted to 1X 10 4 CFU/mL with Luria-Bertani medium. The same amount of bacterial liquid was inoculated into the coated plates and after incubation at 37 ℃ for 24 hours, the change in bacterial numbers was determined.
Statistical analysis all tabular drawings and statistical data were performed using GRAPHPAD PRISM 9.0.0 software. Each experiment was repeated three times and all data are expressed as mean ± standard error. Comparing the two groups using Student t test, the P <0.05 difference was considered statistically significant.
2. Test results
Biocompatibility and antibacterial Activity of ZIF-8-CeO 2 -miR200c Hacat cells and ZIF-8-CeO 2 -miR200c were co-cultured for 7 days, and subsequent FITC/DAPI staining showed no significant difference in cell morphology from the normal group, and the nuclei and cytoplasm were intact (FIG. 3A). The CCK-8 method showed that on day 3, after 3Gy irradiation, the OD of the HaCaT cells of groups B and C decreased, indicating that the irradiation significantly inhibited cell growth (FIG. 3B). On day 6, the OD recovery was significantly higher for group C than for group B (p < 0.05), indicating that ZIF-8-CeO2-miR200C might not affect growth of irradiated cells in the short term, whereas the growth curve of irradiated cells was restored to normal for the long term (> 6 d). As shown in fig. 3C, methicillin-resistant staphylococcus aureus (MRSA, gram positive, g+) and escherichia coli (G-) grew well in the control group, but ZIF-8-CeO2-miR200C effectively inhibited bacterial growth (fig. 3C and 3D), and the bacteriostatic effect was ZIF-8-CeO 2 -miR200C group > ZIF8-CeO 2 group > ZIF8-miR200C group.
In vitro cytological study of ZIF-8-CeO 2 -miR200c the effect of ZIF-8-CeO 2 -miR129 on HUVEC tube formation is shown in FIGS. 4A and 4B. The angiogenesis is obviously limited after the B group is irradiated by 3 Gy, the C group ZIF-8-CeO 2 -miR200C has the best angiogenesis effect (p is less than 0.05), and the angiogenesis effect is ZIF-8-CeO 2 -miR200C group > ZIF 8-CeO 2 group.
HaCaT cells were co-cultured with each set of materials for about 24 hours, and then subjected to a cell scratch test (fig. 4C and 4D). Group B24 h showed a significant decrease in mobility (p < 0.05), which may be associated with the effect of radiation on cell migration, but group C ZIF-8-CeO2-miR200C showed a significant decrease in migration inhibition, indicating that the material increased the mobility of the irradiated cells (p < 0.05), with cell migration effects of ZIF-8-CeO 2 -miR200C > ZIF 8-CeO 2.
Test example 2 in vivo cytological test
RISI mouse model approximately 0.2ml of pentobarbital was intraperitoneally injected to anesthetize each mouse. Then, a linear accelerator (irradiation field 1 cm. Times.1 cm; one irradiation; 30Gy;10min; dose 300 cGy/min) emitting 6 Mev electron beams was used. In this case, the source skin is kept at a distance of 1 meter and the lead plate is used to shield the remaining skin. After irradiation of each group, the irradiated areas were still applied at 48 hour intervals for a total duration of 7-14 days and divided into five groups, each group containing five mice. The five groups were A-E (healthy mice), B (mice + Gy irradiation), C (mice + Gy irradiation +ZIF-8-CeO 2 -miR 200C), D (mice + Gy irradiation +ZIF-8-miR 200C) and E (mice + Gy irradiation +ZIF-8-CeO 2).
To determine the recovery activity, the wound area before/after recovery was examined with IPP6.0 as follows:
Wound recovery case= (initial wound area-wound area after fixed time recovery)/initial wound area x 100%.
The rili score was then calculated using the Douglas and Fowler methods.
Hematoxylin-eosin (HE) staining and histological-based analysis wound samples of each mouse were collected 14 days post-traumatic to prepare paraffin sections, which were then HE stained before obtaining high quality images for data set results.
As shown in fig. 5A, all mice were shaved, photographed, and treated on day 1. On day 14, mice were photographed again and finally local skin tissues were collected for HE staining. The wound recovery of group C, group D and group E was enhanced (p < 0.05) compared to group B, the wound recovery of group C was significantly improved (p <0.05, FIGS. 5B and 5C) compared to group D and group E, and the wound recovery of group D was better than that of group E, indicating that miR200C had the effect of repairing RISI. The result shows that ZIF-8-CeO2-miR200c enhances migration and proliferation activity of epidermal cells so as to induce wound recovery.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (8)
- The application of ZIF-8-CeO 2 -miR200c nano-enzyme in preparing a radioactive skin injury wound repair product is characterized in that the ZIF-8-CeO 2 -miR200c nano-enzyme is prepared by the following method:(1) Dispersing CeO 2 nano particles in deionized water to obtain CeO 2 dispersion, dissolving miR200c in the deionized water to obtain miR200c solution, adding miR200c solution into CeO 2 dispersion, stirring, then adding 2-methylimidazole solution, continuously stirring, then adding zinc nitrate solution for reaction, centrifuging and washing precipitate after the reaction is finished;(2) Dissolving C18PMH-PEG in ultrapure water, adding the precipitate obtained in the step (1), performing ultrasonic treatment in ice bath, and centrifuging to obtain the ZIF-8-CeO 2 -miR200C nano enzyme.
- 2. The use according to claim 1, wherein in step (1) the concentration of the CeO 2 dispersion is 10mg/mL and the concentration of the miR200c solution is 20OD/mL.
- 3. The use according to claim 1, wherein in step (1) the concentration of the 2-methylimidazole solution is 20mg/ml and the concentration of the zinc nitrate solution is 100mg/ml.
- 4. The use according to claim 1, wherein in step (1), the volume ratio of CeO 2 dispersion, miR200c solution, 2-methylimidazole solution and zinc nitrate solution is 1:1:1:2.
- 5. The use according to claim 1, wherein in step (1) the reaction time is 30min.
- 6. The method according to claim 1, wherein in the step (1), the CeO 2 nano-particles are prepared by mixing a cerium nitrate solution and polyallylamine hydrochloride at room temperature, and obtaining CeO 2 NP after centrifugal washing.
- 7. The use according to claim 1, wherein in step (2) the ratio of the addition of C18PMH-PEG, ultrapure water and precipitate is 100mg to 5ml to 10mg.
- 8. The use according to claim 1, wherein in step (2), the time of the ultrasound is 90min, the power is 1000w, and the centrifugation is 21000g for 3h.
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