CN113499474B

CN113499474B - ZIF-67 modified hollow vanadium dioxide shell-core structure micro-nano composite and preparation method and application thereof

Info

Publication number: CN113499474B
Application number: CN202110601363.0A
Authority: CN
Inventors: 彭丽华; 王毛泽; 黄奕谕
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-05-31
Filing date: 2021-05-31
Publication date: 2022-04-12
Anticipated expiration: 2041-05-31
Also published as: CN113499474A

Abstract

The invention discloses a metal-organic ligand framework ZIF-67 modified hollow vanadium dioxide shell-core structure micro-nano composite and a preparation method and application thereof. The multifunctional micro-nano composite with the hollow vanadium dioxide shell-core structure modified by the metal-organic ligand framework ZIF-67 has the advantages of good biocompatibility, low toxicity,does not cause any adverse reaction to organs and has the advantages of antibiosis and angiogenesis promotion. The micro-nano compound provided by the invention and single nano VO₂Compared with the traditional Chinese medicine composition, the traditional Chinese medicine composition has more excellent antibacterial performance and catalytic activity and lower cytotoxicity, has the functions of promoting angiogenesis and accelerating the healing of infected wounds, and can be applied to the repair and regeneration of infected tissues.

Description

ZIF-67 modified hollow vanadium dioxide shell-core structure micro-nano composite and preparation method and application thereof

Technical Field

The invention belongs to the field of biological materials, and particularly relates to a metal-organic ligand framework ZIF-67 modified hollow vanadium dioxide shell-core structure micro-nano composite, and a preparation method and application thereof.

Background

Transition metal chalcogenides (TMDCs) are a new type of two-dimensional nanomaterials after the development of graphene nanomaterials, have excellent chemical stability, extremely large specific surface area and unique biological properties, and are widely studied in biomedical applications. The general chemical formula of TMDCs is MX₂Wherein M is a transition metal atom such as Mo or V; x denotes chalcogen elements such as O, S and Se. For example, MoS₂The nano-sheet has wide application in biological detection, biological imaging, targeted therapy and other biological fields, and TiO₂Has unique biological characteristics in the aspect of tissue regeneration. Vanadium dioxide, a member of TMDCs, is a novel bioactive agent, has excellent enzyme-like activity, and can be used for resisting bacteria, relieving inflammation, regulating cell proliferation and the like. Thus, vanadium dioxide is a very promising tissue regeneration biomaterial, which has not been reported yet.

The surface functional modification of TMDCs has very important influence on the behavior of TMDCs in and out of organisms. TMDCs, although exhibiting their unique physical and chemical properties, still have problems of insufficient biological stability in physiological environments. The surface of the TMDCs is functionally modified, so that the biocompatibility of the TMDCs is improved, the biodistribution of the TMDCs is adjusted, the biotoxicity of the TMDCs is reduced, and the application of the TMDCs in the biomedical field is expanded. The surface modification of TMDCs mainly comprises physical modification and chemical modification. The maximum specific surface area of the TMDCs can generate physical adsorption such as electrostatic adsorption, hydrophobic force, van der Waals force and the like to achieve physical modification; the organic molecules can form C-S chemical bond bonding with the surface atoms of the TMDCs to realize chemical modification.

Zeolite imidazole framework materials (ZIFs) are novel zeolite metal-organic framework materials formed by complexing transition metal ions and imidazole ligands, and have many excellent characteristics of MOFs and high stability and conductivity of traditional zeolites. The skeleton structure of ZIF-67 is made of Co²⁺Ions and N atoms in 2-methylimidazole are hybridized to form a polyhedral structure unit. The cobalt ions released by the compound in an acidic environment can effectively inhibit bacterial activity, and in addition, the cobalt ions can be used as a chemical inducer of hypoxia inducible factor (HIF-1 alpha) to promote the polarization, migration, homing and angiogenesis of endothelial cells. The membrane-core (membrane-core) hybrid structure is formed by chemical bonds and/or physical interactions between inner and outer membranes, endows the membrane-core structured nanocomposites (membrane-core structured nanocomposites) with unique physical, chemical and biological properties, and has huge application potential in intelligent drug delivery.

Wound infection is the most important interference factor in the wound healing process, and exudate is much after infection, so that the local tension of the wound is increased, and even the wound is cracked. For infected wounds, suturing is not possible, drainage should be early, and repair can only be performed if infection is controlled. Therefore, anti-infection, anti-sterilization of infected tissues is one of the key components in the wound healing process. Most of the relevant pathogens require a pH above 6 and their growth is inhibited by the lower pH. Pathogenic bacteria are metabolized in the infected tissue, resulting in a deficiency of nutrients and oxygen in the infected tissue, resulting in increased glycolysis, resulting in an increase in lactate and PaCO2, a decrease in pH, and a shift in pH to an acidic environment due to the body's innate defenses, such as neutrophils, against bacteria from the wound. Under an acidic environment, the film structure ZIF-67 can respond to the acidic environment to release Co ions and has a certain antibacterial function. The acid response effect of ZIF-67 has not been reported previously.

In the case of tissue infections, the body loses the ability to vascularize resulting in tissue ischemia, fibrosis and even necrosis due to insufficient blood supply. Angiogenesis is a crucial stage in the healing process. With newbornThe formation of blood vessels can provide sufficient oxygen, nutrients and growth factors for the damaged tissues to accelerate healing. Therefore, angiogenesis is a necessary condition for the recovery function of damaged tissues. In the angiogenesis process, the migration of endothelial cells under the action of chemokines such as basic fibroblast growth factor (bFGF), Vascular Endothelial Growth Factor (VEGF), uPA and the like is a key link. Co²⁺The ions can stimulate hypoxic conditions by binding directly to HIF1- α and stabilizing HIF1- α, resulting in a synergistic acceleration of angiogenesis-related gene expression, thereby promoting the polarization, migration, homing and neovascularization of endothelial cells.

Disclosure of Invention

Aiming at the content, the invention provides a multifunctional micro-nano composite with a hollow vanadium dioxide shell-core structure modified by a metal-organic ligand framework ZIF-67, which is applied to the antibiosis and angiogenesis of infected tissues. The micro-nano compound has the functions of resisting bacteria and promoting angiogenesis, and has the advantages of good biocompatibility, low toxicity, no adverse reaction on organs and the like.

The invention provides a preparation method of a metal-organic ligand framework ZIF-67 modified hollow vanadium dioxide shell-core structure micro-nano composite, which comprises the following steps:

(1) will V₂O₅Adding into oxalic acid solution, stirring to form suspension, placing into a hydrothermal reaction kettle, and preparing to obtain VO through hydrothermal reaction₂；

(2) The prepared VO₂Uniformly dispersing the mixture in a methanol solution, adding the methanol solution of cobalt nitrate hexahydrate and 2-methylimidazole, stirring at room temperature until the mixture fully reacts, and drying in vacuum to obtain the ZIF-67-coated hollow vanadium dioxide shell-core structure micro-nano composite material.

Further, V described in step (1)₂O₅The mol ratio of the oxalic acid to the oxalic acid is 1: (1-150), wherein the concentration of the oxalic acid solution is 0.6-5 mol/L.

Furthermore, the hydrothermal reaction temperature is 120-300 ℃, and the reaction time is 6-48 h.

Further, in the step (2), VO₂Cobalt nitrate hexahydrateAnd the feeding mass ratio of 2-methylimidazole is 1: (2-60): (4-90).

Further, in the step (2), the vacuum drying temperature is 40-80 ℃.

The invention also provides a ZIF-67 modified hollow vanadium dioxide shell-core structure micro-nano composite, which is characterized in that the micro-nano composite is of a core-membrane double-layer structure with the ZIF-67 coated on the surface of vanadium dioxide, and the vanadium dioxide is of a hollow structure.

The invention further provides various applications of the ZIF-67 modified hollow vanadium dioxide shell-core structure micro-nano composite, such as application in preparing medicines for treating tissue infection; the application in the preparation of antibacterial drugs and/or angiogenesis promoting drugs for infected tissues; application in preparing wound healing medicine.

The invention has the advantages that: the multifunctional micro-nano composite with the hollow vanadium dioxide shell-core structure modified by the metal-organic ligand framework ZIF-67 has the advantages of good biocompatibility, low toxicity, no adverse reaction on organs, antibiosis and angiogenesis promotion. And single nano VO₂Compared with the prior art, the composite material has more excellent antibacterial performance and catalytic activity, and meanwhile, the cobalt ions released under the acidic condition can promote angiogenesis.

Drawings

The invention will be further described with reference to the accompanying drawings;

FIG. 1 is a microscope observation image of a metal-organic ligand framework ZIF-67 modified hollow vanadium dioxide core-membrane structure micro-nano composite synthesized in example 1; wherein a and b are compound scanning electron micrographs, c is a scanning picture of the compound under an element scanning electron microscope, d is a vanadium element scanning picture, e is a cobalt element scanning picture, and f illustrates that ZIF-67 is completely coated on the surface of the vanadium dioxide core.

FIG. 2 is a UV-Vis spectrum measured by different sets of TMB methods of example 3;

FIG. 3 is a statistical plot of the concentration of Co ions released from the composite structure under different pH conditions according to example 4;

FIG. 4 shows the growth of colonies from different groups of example 5;

FIG. 5 shows the angiogenesis in example 6 under different pH conditions;

FIG. 6 is a statistical graph showing the survival rate of cells of each group at different concentrations in example 7;

FIG. 7 shows H & E staining of organs at different concentrations in example 8.

Detailed Description

The invention is further illustrated by the following examples.

Example 1 Synthesis of hollow vanadium dioxide core-membrane structure micro-nano composite modified by metal-organic ligand framework ZIF-67

1.82g V₂O₅Dissolving the mixture in 450ml of 1.6 mol/L oxalic acid solution, and stirring the mixed solution at room temperature until the solution becomes an earthy yellow suspension. And (3) placing the obtained suspension in a polytetrafluoroethylene reaction kettle for hydrothermal reaction, and keeping the temperature at 180 ℃ for 36 hours. Naturally cooling to normal temperature after the reaction is finished and collecting blue-black precipitate VO₂Drying in a constant temperature ventilation drying oven at 60 ℃.

100mg of the prepared VO was taken out₂Uniformly dispersing in 200mL of methanol solution, adding 3g of cobalt nitrate hexahydrate, uniformly stirring, adding 300mL of 0.2g/mL of 2-methylimidazole methanol solution, stirring at normal temperature for more than 24 hours to allow the mixture to fully react, centrifugally washing the prepared sample by using deionized water and ethanol, and drying in vacuum at 60 ℃ to obtain VO coated with ZIF-67₂Nanocomposite, hereinafter example referred to as VO₂@ ZIF-67. Observed by SEM and TEM images, as shown in fig. 1.

The experimental results are as follows: the prepared vanadium dioxide is of a hollow structure, and the ZIF-67 is coated on the surface of the vanadium dioxide to form a core-film double-layer structure.

Example 2 Synthesis of hollow vanadium dioxide core-membrane structure micro-nano composite modified by metal-organic ligand framework ZIF-67

1.82g V₂O₅Dissolved in 500ml of 1.2 mol/L oxalic acid solution, and the mixed solution is stirred at room temperature until the solution becomes a yellowish brown suspension. And (3) placing the obtained suspension in a polytetrafluoroethylene reaction kettle for hydrothermal reaction, and keeping the temperature at 300 ℃ for 28 h. After the reaction is finishedNaturally cooling to normal temperature and collecting blue-black precipitate VO₂Drying in a constant temperature ventilation drying oven at 60 ℃.

100mg of the prepared VO was taken out₂Uniformly dispersing in 300mL of methanol solution, adding 5g of cobalt nitrate hexahydrate, uniformly stirring, adding 500mL of 0.15g/mL of 2-methylimidazole methanol solution, stirring at normal temperature for more than 24 hours to allow the mixture to fully react, centrifugally washing the prepared sample by using deionized water and ethanol, and drying in vacuum at 80 ℃ to obtain VO coated with ZIF-67₂Nanocomposite, hereinafter example referred to as VO₂@ ZIF-67. The composite material is also detected to have a core-membrane double-layer structure of ZIF-67 coated hollow vanadium dioxide.

Example 3 Nanolase function of peroxidase-like Activity

2mL of Na having a pH of 4.8₂HPO₄(0.2mol/L) -citric acid (0.1mol/L) buffer solution was mixed with each of the following a-f groups of samples (a)0.48mg of VO₂@ZIF-67+0.05mL 0.02mol/LTMB+0.3mL 0.6mmol/LH₂O₂；(b)0.48mg VO₂+0.05mL 0.02mol/L TMB+0.3mL0.6mmol/LH₂O₂；(c)0.48mg VO₂+0.3mL 0.6mmol/LH₂O₂；(d)0.48mg VO₂@ ZIF-67+0.05mL of 0.02mol/L TMB; (e)0.05mL of 0.02mol/L TMB and 0.3mL0.6mmol/LH₂O₂；(f)0.48mg ZIF-67+0.05mL 0.02mol/L TMB+0.3mL 0.6mmol/LH₂O₂(ii) a After mixing well, 0.05mL of 1.0X 10 was added^-3And (5) mol/L enzyme solution to be detected. The mixture was reacted for 3.5min and the UV-Vis spectra of each group were determined. With H₂O₂+ TMB is the control group, and the control experiment is performed with the rest of the groups as experimental groups.

The experimental results are shown in fig. 2: the composite structure is superior to a separation unit in simulating the catalytic activity of peroxidase, is similar to a cascade amplification effect in organisms, and shows that the catalytic activity of the core is further improved by a membrane layer structure due to the larger porosity, the higher specific surface area and the larger pore volume of a core-membrane double-layer structure.

Example 4 acid-responsive Release Effect of composite structures

Using inductively coupled plasmaThe initial concentration of the sample is 1.0 × 10 as determined by an emission spectrometer (ICP-OES PE optima8000)^-3VO prepared in example 1 at different pH solutions (pH 5.4, 6.8, 7.4) and different times (t 0h, 1h, 12h, 24h, 36h, 48h) in mol/L₂@ ZIF-67 Co²⁺The cumulative amount of release of (a).

The experimental results are shown in fig. 3: the lower the pH value of the membrane structure ZIF-67 is under three different pH conditions, the higher the Co ion release amount is, and the membrane structure ZIF-67 has an acid response effect; under three different pH conditions, Co ion release was rapid within the first 1h, followed by gradual stabilization, indicating that the Co ion release concentration would be maintained at a sustained level to maintain long-term effect. In conclusion, the acid response effect of the membrane structure ZIF-67 has great application potential in the treatment of wound infection.

EXAMPLE 5 antibacterial function of composite Structure

Single colonies of non-drug-resistant bacteria and drug-resistant bacteria were inoculated on solid LB medium, and 50mL of sterile liquid LB medium (containing bacto tryptone (0.5g), bacto yeast extract (0.25g), and NaCl (0.5 g)). Then suspending the non-drug-resistant bacteria and drug-resistant bacteria at 37 deg.C for 180 rpm^-1The cells were incubated overnight on a rotary shaker. Subsequently, the bacteria were diluted to 1 × 10 with sterile Phosphate Buffered Saline (PBS)⁶Colony Forming Unit (CFU) mL^-1. The resulting bacterial solution (200. mu.L) was mixed with 200. mu.g/mLVO₂@ ZIF-67 and 50. mu. mol H₂O₂At 37 ℃ for 180 rpm^-1The rotary vibrating screen of (1) was thoroughly mixed for 4 hours. After the treatment, the cells were cultured overnight in LB medium at 37 ℃ and the number of colonies was counted by the CFU method. PBS as blank control, and VO₂And VO₂@ ZIF-67 control experiments were performed.

The results of the experiment are shown in FIG. 4: compared with a blank group and a VO2 group, the antibacterial performance of the VO2@ ZIF-67 group is remarkably improved, which shows that the antibacterial performance of vanadium dioxide is enhanced by ZIF-67 modification; the ZIF-67 releases cobalt ions under acidic conditions due to the acidic environment caused by bacterial life, which shows that the efficient antibacterial performance of the composite material is the synergistic effect of the enzymatic activity of the cobalt ions.

EXAMPLE 6 angiogenesis function of composite Structure

Aliquots (200 μ L) of growth factor reduced matrix gel base membrane matrix were added to individual wells of 48-well cell culture plates and polymerized for 30 minutes at 37 ℃. Under different conditions (VO)₂@ZIF-67、VO₂@ZIF-67/pH5.4、VO₂@ZIF-67/pH6.8、VO₂@ ZIF-67/pH7.4, concentration 200. mu.g/mL) of human umbilical vein endothelial cells at 1X 10 per well⁴Density seeding of cells. The blocked vascular network was observed microscopically at different time points (t ═ 4, 6, 12, 24h) of incubation at 37 ℃. Tube formation was quantified by measuring tubule length in four randomly selected regions per well, and the values from three independent experiments were averaged and shown as mean ± standard deviation.

The results of the experiment are shown in FIG. 5: due to the ZIF-67 acid response effect of the membrane structure, the micro-nano compound can release cobalt ions under an acidic condition, and effectively promotes angiogenesis.

Example 7 cytotoxicity

Culturing human umbilical vein endothelial cells labeled with luciferase, transferring into 48-well plate, and dividing into 5 groups, shown in FIG. 4, which are blank control group and VO₂Group, VO₂@ ZIF-67/pH5.4 group, VO₂@ ZIF-67/pH6.8 group, VO₂@ ZIF-67/pH7.4 group, wherein each group was divided into 10 subgroups, and the concentrations were 0, 2.5, 5, 10, 20, 25, 50, 75, and 100. mu.g/mL, respectively. Each set was provided with 3 multiple wells. Cell density of 1X 10⁵And culturing for 24h per mL.

The detection is carried out by a CCK-8 kit method. And (3) measuring the absorbance value by adopting an enzyme-linked immunosorbent assay detector under the condition that the wavelength is 490nm, and calculating the cell survival rate by taking the untreated cells as reference. The cell viability was calculated as follows:

cell survival (%) ═ OD₄₉₀(sample)/OD₄₉₀(control). times.100%; wherein, OD₄₉₀(sample) OD value, OD of experimental group₄₉₀(control) is the OD value of the blank control group.

The experimental results are shown in fig. 6: the vanadium dioxide has certain cytotoxicity, and the membrane modification of the ZIF-67 can reduce the cytotoxicity of the hollow vanadium dioxide, which shows that the biological safety of the hollow vanadium oxide is improved by the membrane modification of the ZIF-67.

Example 8 Biosafety

SD rats were randomly divided into control group, VO₂@ ZIF-6750. mu.g/kg group, VO₂@ ZIF-67100. mu.g/kg group and VO₂@ ZIF-67200. mu.g/kg group. First, rats were anesthetized by intraperitoneal injection of 3% pentobarbital (1mL/kg) prior to surgery and placed on a sterile drape to provide sterile conditions during surgery. The backs of the rats were shaved, depilated and disinfected, and then 15mm long squares of skin were excised. Each wound of each group was then infected with e. The dosage is 10⁷CFU/wound, combined PBS (control), VO₂@ZIF-67 50μg/kg、VO₂@ ZIF-67100. mu.g/kg and VO₂@ ZIF-67200. mu.g/kg of treatment. The wound was then covered with a nonwoven and secured with a surgical adhesive. To evaluate VO₂@ ZIF-67 therapeutic Effect on wound infection bacterial counts at various time points (days 3, 7, 11) were measured by plate diffusion method and regularly observed and photographed. Rats were sacrificed and wound skin tissue was taken, fixed with 4% paraformaldehyde solution and embedded in paraffin. H was carried out on

days

3, 7, 11 and 20, respectively&And E, dyeing. To evaluate biosafety in vivo, major organs (heart, liver, spleen, lung and kidney) were subjected to H on day 20&And E, dyeing.

The results of the experiment are shown in FIG. 7: even under the condition of high concentration in vivo, the micro-nano compound does not cause any adverse reaction to organs, and the biological safety of the ZIF-67 modified hollow vanadium dioxide shell-core structure micro-nano compound is good.

Claims

1. A preparation method of a metal-organic ligand framework ZIF-67 modified hollow vanadium dioxide shell-core structure micro-nano composite is characterized by comprising the following steps:

(2) The prepared VO₂Uniformly dispersing in methanol solution, adding hexaAnd (3) mixing the cobalt nitrate hydrate and the methanol solution of 2-methylimidazole at room temperature to fully react, and drying in vacuum to obtain the ZIF-67-coated hollow vanadium dioxide shell-core structure micro-nano composite material.

2. The method according to claim 1, wherein V in the step (1)₂O₅The mol ratio of the oxalic acid to the oxalic acid is 1: (1-150), wherein the concentration of the oxalic acid solution is 0.6-5 mol/L.

3. The preparation method according to claim 1, wherein in the step (1), the hydrothermal reaction temperature is 120 to 300 ℃ and the reaction time is 6 to 48 hours.

4. The method according to claim 1, wherein in step (2), VO is produced₂The feeding mass ratio of the cobalt nitrate hexahydrate to the 2-methylimidazole is 1: (2-60): (4-90).

5. The method according to claim 1, wherein in the step (2), the vacuum drying temperature is 40 to 80 ℃.

6. The ZIF-67 modified hollow vanadium dioxide shell-core structure micro-nano composite prepared by the method of any one of claims 1 to 5, characterized in that the micro-nano composite is a core-membrane double-layer structure with ZIF-67 coated on the surface of vanadium dioxide, and the vanadium dioxide is of a hollow structure.

7. The use of the ZIF-67 modified hollow vanadium dioxide shell-core structure micro-nano complex of claim 6 in the preparation of a medicament for the treatment of tissue infection.

8. The application of the ZIF-67 modified hollow vanadium dioxide shell-core structure micro-nano composite in preparation of antibacterial drugs and/or angiogenesis promoting drugs for infected tissues.

9. The application of the ZIF-67 modified hollow vanadium dioxide shell-core structure micro-nano composite in preparation of a wound healing drug.