CN115400226A

CN115400226A - ZIF-8@ Rutin nano composite material with antibacterial and anti-inflammatory functions as well as preparation method and application thereof

Info

Publication number: CN115400226A
Application number: CN202211043003.4A
Authority: CN
Inventors: 李雪; 夏晓敏; 王楠; 孙振龙; 闫顺杰; 李佃华
Original assignee: Weigaozhi Interventional Medical Device Technology Shandong Co ltd; Weigao Holding Co ltd; Qingdao University
Current assignee: Weigaozhi Interventional Medical Device Technology Shandong Co ltd; Weigao Holding Co ltd; Qingdao University
Priority date: 2022-08-29
Filing date: 2022-08-29
Publication date: 2022-11-29

Abstract

The invention provides a ZIF-8@ Rutin nano composite material with antibacterial and anti-inflammatory functions, which is prepared from ZIF-8 nano particles and rutin. The invention adopts ZIF-8 nano-particles as carriers, wherein Zn ²⁺ Metallo-organic ligands as ZIF-8 also play a key role in the healing of local wounds: wounds deficient in zinc (Zn) often experience slower healing times. Zinc also acts as a cofactor for metalloproteins and plays an important role in the regeneration of extracellular matrix by enhancing re-epithelialization. Compared with metallic silver, the metallic zinc is easier to obtain and has lower cost. Meanwhile, the natural anti-inflammatory drug rutin separated from the plants can eliminate excessive active oxygen released by ZIF-8 nano particles, reduce inflammatory reaction, promote wound growth and promote the wound healing process.

Description

ZIF-8@ Rutin nano composite material with antibacterial and anti-inflammatory functions as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a ZIF-8@ Rutin nano composite material with antibacterial and anti-inflammatory functions, and a preparation method and application thereof.

Background

Currently, wound infection is a common problem facing clinically in the international medical field. Wound healing is a complex, highly regulated biological process, and in healthy individuals, a small incisional body often promotes wound healing by tightly coordinating cell migration, innervation, and angiogenesis at a level of inflammation. But can delay the healing of wounds under the action of factors which cannot be resisted by the outside, such as the aging change of the body, and the occurrence of obesity, diabetes and cardiovascular diseases. Importantly, the greatest challenge facing wound healing is wound infection, and the use of antibiotics is an important measure in the prevention and treatment of wound infection. However, the unreasonable application of antibiotics can produce some side effects, such as anaphylaxis caused by systemic administration, gastrointestinal reaction and other adverse reactions, and most fatally, the occurrence of drug-resistant bacteria improves the treatment of infected wounds to another height, consumes medical resources and increases the pain of patients. Currently, antibiotics are still being used and abused clinically, thereby endangering human health. Therefore, it is very important and valuable to develop a novel drug which does not have side effects and does not develop drug resistance.

The nano material nano enzyme is concerned by people in the antibacterial field. Zeolite imidazolate framework-8 (ZIF-8) is composed of ligand 2-methylimidazole and transition metal ion Zn ²⁺ A dodecahedral complex formed by self-assembly. ZIF-8 is widely applied to the fields of catalysis, adsorption, particularly drug delivery and the like by virtue of the excellent characteristics of regular pore channel structure, large specific surface area, chemical stability, convenience in modification, simplicity in synthesis and the like. An important reason why ZIF-8 is widely used in antibacterial therapy is that ZIF-8 is unstable in aqueous solution and undergoes hydrolysis reaction to release Zn ions to exert bactericidal activity. It is known that the organic ligand metal ions in the metal nano material release Reactive Oxygen Species (ROS) to make the metal nano material have certain cytotoxicity, and the Reactive Oxygen Species (ROS) as a cell secondary messenger of inflammatory cytokine signals can induce the aggravation of local inflammatory reaction and delay the healing of wounds. In recent years, many studies have been made on loading various antibacterial drugs on ZIF-8 as a drug carrier, but there is no report on a drug which can remove excessive active oxygen released by ZIF-8 during sterilization, reduce cytotoxicity, and have anti-inflammatory properties. Therefore, it is a problem to be solved how to make ZIF-8 have anti-inflammatory properties while exerting in vivo antibacterial properties.

Rutin is a natural flavonoid compound, and has wide important pharmacological functions, including anticancer, anti-inflammatory, antibacterial and antioxidant functions. The excellent antioxidant activity of rutin can remove redundant ROS released during ZIF-8 structure collapse, so that the damage to cells is reduced, but the solubility of rutin limits the bioavailability, and numerous reports of rutin carrier wrapping are reported, so that how to combine rutin and ZIF-8 composite nanoparticles and simultaneously have the antibacterial and anti-inflammatory properties is not reported in the research at present.

Therefore, those skilled in the art have made efforts to develop a new preparation method of ZIF-8 composite nanoparticles and can load functional materials suitable for antibacterial and anti-inflammatory therapy.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a ZIF-8@ rutin nanocomposite material having both antibacterial and anti-inflammatory functions, and a preparation method and an application thereof, and the nanocomposite material provided by the present invention can release effective molecules to exert antibacterial and anti-inflammatory effects through the collapse of a ZIF-8 structure in a local infected wound, so as to reduce the occurrence of infection and promote the healing of the wound.

The invention provides a ZIF-8@ Rutin nano composite material with antibacterial and anti-inflammatory functions, which is prepared from ZIF-8 nano particles and rutin.

Preferably, the mass ratio of the ZIF-8 nanoparticles to the rutin is 1.

Preferably, the particle size of the ZIF-8 nano-particles is 40 +/-3 nm, and the particle size of the ZIF-8@ Rutin nano-composite material is 62 +/-3 nm.

Preferably, the ZIF-8 nanoparticles have a dodecahedral morphology; the ZIF-8@ Rutin nano composite material is a secondary particle formed by agglomerating primary particles.

The invention also provides a preparation method of the nano composite material, which comprises the following steps:

a) Under a closed condition, mixing and stirring ZIF-8 nano-particles and rutin in an organic solvent, and reacting to obtain a reaction product;

or under a closed condition, adding rutin in the process of preparing the ZIF-8 nano particles, mixing and stirring, and reacting to obtain a reaction product;

b) Under a closed condition, standing the reaction product under a heating condition, and then performing centrifugal separation to obtain a crude product;

c) And washing the crude product, drying and crushing to obtain the ZIF-8@ Rutin nano composite material.

Preferably, the ZIF-8 nanoparticles are prepared as follows:

1) Respectively dissolving zinc nitrate hexahydrate and 2-methylimidazole in methanol to obtain a zinc ion methanol solution and a 2-methylimidazole methanol solution;

2) And mixing and stirring the zinc ion methanol solution and the 2-methylimidazole methanol solution, standing, centrifuging, washing and drying to obtain white powdery ZIF-8.

Preferably, the mass ratio of the ZIF-8 nanoparticles to the rutin is 1.

Preferably, the organic solvent is selected from methanol.

Preferably, the heating condition is at a temperature of 50 ℃ to 55 ℃.

The invention also provides application of the nano composite material in preparation of a medicine with antibacterial and anti-inflammatory effects and capable of promoting wound healing.

Compared with the prior art, the invention provides the ZIF-8@ Rutin nano composite material with the antibacterial and anti-inflammatory functions, which is prepared from ZIF-8 nano particles and rutin. The invention adopts ZIF-8 nano-particles as carriers, wherein Zn ²⁺ The metal organic ligand as ZIF-8 also plays a key role in healing of local wounds: zinc (Zn) -deficient wounds typically experience slower healing times. Zinc also acts as a cofactor for metalloproteins and plays an important role in the regeneration of extracellular matrix by enhancing re-epithelialization. Compared with metallic silver, the metallic zinc is easier to obtain and has lower cost. Meanwhile, the natural anti-inflammatory rutin separated from the plants can eliminate excessive active oxygen released by the ZIF-8 nano particles, reduce inflammatory reaction, promote wound growth and promote the wound healing process.

Drawings

FIG. 1 is a Scanning (SEM) electron micrograph of ZIF-8;

FIG. 2 is a Transmission Electron (TEM) micrograph of ZIF-8;

FIG. 3 is a particle distribution diagram of ZIF-8;

FIG. 4 is a transmission electron micrograph of synthetic ZIF-8@ RUTIN nanoparticles;

FIG. 5 is a particle distribution plot of synthesized ZIF-8@ RUTIN nanoparticles;

FIG. 6 is an XRD pattern of ZIF-8 and ZIF-8@ RUTIN nanoparticles;

FIG. 7 is an FTIR plot of ZIF-8 and ZIF-8@ RUTIN nanoparticles;

FIG. 8 is ZIF-8@ Rutin (30. Mu.g mL) ^-1 ) Rutin (40. Mu.g mL) ^-1 ) And ZIF-8@ Rutin (40. Mu.g mL) ^-1 ) Inhibitory behavior on the metabolic activity of escherichia coli and staphylococcus aureus within 24 hours;

FIG. 9 is a CFU count histogram for a single biofilm of E.coli and S.aureus;

FIG. 10 is a transmission electron micrograph of the biofilm structure of two bacteria;

FIG. 11 is a graph of inhibition of macrophage inflammation for ZIF-8@ RUTIN nanoparticles;

FIG. 12 is the experimental picture of in vivo antibacterial anti-inflammatory animal by ZIF-8@ RUTIN nanoparticles.

Detailed Description

In the invention, the mass ratio of the ZIF-8 nanoparticles to the rutin is 1.

The particle size of the ZIF-8 nano particles is 40 +/-3 nm, and the particle size of the ZIF-8@ Rutin nano composite material is 62 +/-3 nm.

The ZIF-8 nanoparticles have a dodecahedral morphology; the ZIF-8@ Rutin nano composite material is a secondary particle formed by agglomeration of primary particles, and has a flower-like shape.

In the present invention, the ZIF-8 nanoparticles are prepared as follows:

Specifically, the mass ratio of the zinc nitrate hexahydrate to the 2-methylimidazole is 0.74.

In the step 2), the mixing and stirring are carried out under a sealed condition, the mixing and stirring temperature is normal temperature, and the stirring time is 4 hours.

After stirring, standing at 50-55 ℃.

The present invention is not particularly limited to a specific method for the centrifugal separation, and any method known to those skilled in the art may be used.

The washing was methanol washing.

The present invention is not particularly limited to the specific method for drying, and may be any method known to those skilled in the art.

After obtaining the ZIF-8 nano-particles, mixing and stirring the ZIF-8 nano-particles and rutin in an organic solvent under a closed condition, and reacting to obtain a reaction product.

wherein the mass ratio of the ZIF-8 nanoparticles to the rutin is 1. The organic solvent is selected from methanol.

Under a closed condition, standing the reaction product under a heating condition, and then performing centrifugal separation to obtain a crude product; the temperature of the heating condition is 50-55 ℃.

And finally, washing the crude product, drying and crushing to obtain the ZIF-8@ Rutin nano composite material. The present invention is not particularly limited to the specific methods for washing, drying and pulverizing, and may be methods known to those skilled in the art.

The metal ion ligand in the ZIF-8 has positive charge, and has electrostatic adsorption with hydroxyl (-OH) in a Rutin (Rutin) structure, so that the Rutin can be effectively adsorbed on the surface of the ZIF-8 to form the ZIF-8@ Rutin composite nano-particles. Due to the hydrolyzability of ZIF-8, a large amount of Zn is released along with the collapse of the ZIF-8 structure when contacting human body fluid ²⁺ 2-methylimidazole organic ligand and free rutin, and the three simultaneously and synergistically play an antibacterial role. The positive charge carried by the free metal ions increases the lipophilicity of the ZIF-8 material, thereby increasing the compatibility of ZIF-8 with and permeability of bacterial cell membranes to kill bacteria. Furthermore, the effect of electrostatic attraction to the bacterial surface allows the antimicrobial agent to be targeted to the bacterial surface. The combination of these dual effects can effectively improve the antibacterial efficiency. At the same time, zn ²⁺ The release of entrained ROS achieves bactericidal effects by participating in the synthesis of bacterial biofilms and nucleic acid metabolism, as well as other important activities.

The invention has the following technical effects:

(1) According to the invention, an organic solvent phase is used, and a mixed solvent magnetic stirring method is utilized to rapidly and efficiently prepare smaller molecular ZIF-8 nanoparticles with uniform particle size distribution, high crystallinity and regular morphology;

(2) According to the invention, the rutin molecules and metal cations are interacted to increase the functional molecule load capacity through a chemical electrostatic adsorption principle, and the functional molecule load capacity is loaded on the surface of the ZIF-8 nano particles, so that no additional step is needed, the experimental process is convenient and efficient, and the drug load rate ratio is considerable.

(3) By utilizing the unstable characteristic of ZIF-8 in water and along with the collapse of a ZIF-8 carrier structure, the released metal cations form electrostatic adsorption with the surface of bacteria, so that the targeted sterilization effect is achieved. And simultaneously, the anti-inflammatory drug rutin is loaded into the wound, so that the anti-inflammatory effect of eliminating redundant ROS is achieved. The ZIF-8 composite nano-particles are effectively enriched at the infected part in vivo, and the prepared ZIF-8 composite nano-particles can be used for the treatment integration of anti-inflammation, antibiosis and wound healing promotion of local infection in vivo.

In order to further understand the present invention, the following describes the ZIF-8@ rutin nanocomposite material with antibacterial and anti-inflammatory functions, and the preparation method and application thereof, with reference to the following examples, and the scope of the present invention is not limited by the following examples.

Example 1

Preparation of zeolitic imidazolate framework materials (ZIF-8)

1. 0.74g (2.5 mmol) of Zn (NO 3) was weighed ₂ ·6H ₂ O was dissolved in 20mL of methanol and stirred to dissolve it sufficiently.

2. 3.28g (40 mmol) of 2-methylimidazole was weighed out and dissolved in 80ml of methanol, and stirred to be sufficiently dissolved.

3. Zn (NO 3) ₂ ·6H ₂ Mixing the O solution and the 2-methylimidazole solution, sealing the opening of the beaker by using a sealing film, and stirring for 4 hours at normal temperature on a constant-temperature magnetic stirrer.

The mixed solution was allowed to stand at 4.50 ℃ for 1h, and centrifuged at 9500rpm for 15min for separation. Washing the crude product with methanol for three times, drying at 60 ℃ overnight, and grinding to obtain white solid powder ZIF-8.

Preparation of composite nano material ZIF-8@ Rutin

Method (1): ZIF-8: 1 is synthesized by sealing and stirring in methanol for 12h. The mixed solution was sealed and allowed to stand at 50 ℃ for 1h, and centrifuged at 9500rpm for 15min for separation. The crude product was washed three times with methanol, dried overnight under vacuum at 60 ℃ and milled to give the solid yellow powder particles ZIF-8@ RUTIN.

Method (2): 2.5mmol (1.525 g) Rutin is added when ZIF-8 is synthesized, the mixture is sealed and stirred for 4h at normal temperature, the mixed solution is sealed and kept stand for 1h at 50 ℃, and the mixture is centrifuged for 15min at 9500rpm for separation. The crude product was washed three times with methanol, dried overnight under vacuum at 60 ℃ and milled to give the pale yellow solid powder particles of ZIF-8@ RUTIN.

Product characterization and Performance determination

(1) Electron microscope detection

FIGS. 1 and 2 are Scanning (SEM) and Transmission (TEM) electron micrographs of ZIF-8, respectively, showing a uniform distribution of all nanoparticle size particles, 40. + -.3 nm in size (as shown in FIG. 3), and a regular dodecahedron morphology. As shown in FIG. 4, the synthesized ZIF-8@ RUTIN nanoparticles have a flower-like shape under a transmission electron microscope, the particle diameter is obviously larger than that of a ZIF-8 monomolecular particle, about 62 +/-3 nm (FIG. 5), and probably the composite nanoparticles formed by Rutin molecules due to electrostatic adsorption are easy to cluster due to electrostatic adsorption and are adsorbed on the surface of the ZIF-8 particles.

(2) X-ray diffraction (XRD), fourier Infrared Spectroscopy (FTIR)

FIG. 6,7 investigated the structural properties of ZIF-8 and ZIF-8@ rutin nanoparticles by XRD and FTIR analysis. The XRD mode and FTIR of the ZIF-8 are consistent with the previously reported ZIF-8 structure data, which shows that the structure of the original ZIF-8 is not changed after the ZIF-8 is used for loading rutin. The reduction of XRD peak height of ZIF-8@ rutin nanoparticles may be due to the lower degree of crystallization detected after loading rutin than ZIF-8.

From FIG. 7 by infrared spectroscopy at 2943.80 and 2958.68cm ^-1 The absorption peaks at point A belong to the stretching vibration peaks of C-H bonds in methyl and imidazole rings respectively. At 1429.42cm ^-1 The C = N bond stretching vibration peak on the imidazole ring appears at 426.78cm (point B) ^-1 A Zn-N stretching vibration peak appears at point D. No absorption peak ascribed to N-H \8230, N hydrogen bond (2696.86) and N-H bond (1429.42 cm) in 2-methylimidazole were found in our sample ^-1 ) The vibration absorption peak of (2) shows that 2-methylimidazole can be completely deprotonated in the synthesis system, and pure-phase ZIF-8 can be prepared.

(3) The antibacterial performance test of the ZIF-8@ Rutin composite nano-particles comprises the following steps:

respectively incubating a certain concentration of bacteria culture solution (Escherichia coli strain represents gram-negative bacteria and Staphylococcus aureus strain represents gram-positive bacteria) with culture solution (control group) and culture solution (blank group) dispersed with ZIF-8@ Rutin composite nanoparticles for 24h, taking out 10 μ L of each sample, diluting with PBS 10 μ L ⁶ Taking 20 mu L of the diluent, plating the diluent on an LB solid culture medium, and culturing at the constant temperature of 37 ℃ for 12h. And then, recording the number of colonies on the culture plate, and comparing the antibacterial effects of the experimental group and the blank group, wherein the antibacterial properties of the experimental group are all superior to those of the blank group.

ZIF-8@Rutin(30μg mL ^-1 ) Rutin (40. Mu.g mL) ^-1 ) And ZIF-8@ Rutin (40. Mu.g mL) ^-1 ) The inhibitory behavior of the metabolic activity of escherichia coli and staphylococcus aureus over 24 hours and the CFU count statistics of individual biofilms of escherichia coli and staphylococcus aureus are shown in fig. 8 and 9. FIG. 8 is ZIF-8@ Rutin (30. Mu.g mL) ^-1 ) Rutin (40. Mu.g mL) ^-1 ) And ZIF-8@ Rutin (40. Mu.g mL) ^-1 ) Inhibitory behavior on the metabolic activity of escherichia coli and staphylococcus aureus within 24 hours. FIG. 9 is a CFU count histogram of a single biofilm of E.coli and S.aureus. Different letters represent values that are significantly different from each other group (n =6,p<0.05)

As can be seen from the graphs, the CFU count was significantly reduced in the ZIF-8NPs group, and the E.coli group was reduced 3-fold and S.aureus was reduced 6-fold (p < 0.001) for both pathogens. Rutin was significantly less effective as an antibacterial agent than the ZIF-8 group, but was still statistically different compared to the control group. Importantly, although ZIF-8@ Rutin NPs have inferior antibacterial properties compared to ZIF-8NPs, there is still a significant difference in their CFU counts. The antibacterial performance of ZIF-8NPs is slightly reduced due to rutin loading, but the novel ZIF-8@ Rutin NPs still have enough bactericidal capability.

Transmission electron microscopy (fig. 10) visualizes that the biofilm structure of the two bacteria was altered by different materials. Normal s.aureus and e.coli have intact cell walls and smooth cell membranes with sharp edges. In contrast, the bacteria treated with ZIF-8@ Rutin had incomplete membranes, even collapsed, indicating that the ZIF-8@ Rutin composite nanoparticles had excellent bactericidal effects.

(4) In vitro experiment of ZIF-8@ Rutin composite nanoparticles

Cells (L969) with a certain concentration are co-cultured with a culture solution (a control group) and a common culture solution (a blank group) which are respectively dispersed with ZIF-8@ Rutin composite nano particles to detect the cytotoxicity of the ZIF-8@ Rutin. The results showed that ZIF-8@ Rutin was at 40. Mu.g mL ^-1 Has biocompatibility. The ZIF-8 is doped with Rutin, so that the cell activity is obviously improved, and the cytotoxicity is reduced. This is probably due to the antioxidant and ROS-scavenging properties of rutin.

The anti-inflammatory properties of ZIF-8@ Rutin NP were assessed by quantitatively targeting M1 phenotype macrophage-associated cytokines (IL-1 β, IL-6 and TNF- α) and M2 phenotype macrophage-associated cytokines (IL-10, arg-1 and TGF- β) using mouse macrophages (RAW 264.7) as an in vitro model of inflammation. The results showed that ZIF-8@ Rutin inhibited the expression of the factors associated with M1-phenotype macrophages in vivo and promoted the expression of cytokines associated with M2-phenotype macrophages (FIG. 11).

(5) In vivo experiment of ZIF-8@ Rutin composite nanoparticles

By mixing 100. Mu.L of Staphylococcus aureus suspension (1X 10) ⁶ CFU mL-1) was dropped on a full-thickness circular wound of 5mm diameter on the back of the mouse to establish an in vitro model of inflammation. PBS was added dropwise to the back wound of the mouse as a positive control (n = 5). After the inflammation model was successfully established, the infected wound was treated with a dressing of ZIF-8@ Rutin to seal the wound. The healing area of the wound on the back of the mice in different experimental groups was recorded at 0,2,4,6,8, 10d after the establishment of the inflammation model. Compared with the treatment effect of the experimental group and the control group, the healing speed of the back wound of the mice in the experimental group is obviously higher than that of the control group, and the infection symptom disappears. Finally, all tissue surrounding the mouse dorsal wound was excised on day 10, H&The results of E-staining and Masson-staining observed that the re-epithelialization of the tissues, collagen fiber deposition, was significantly greater in the experimental group than in the control group, and inflammatory cell infiltration was significantly less in the control group. Immunohistochemical and immunofluorescent staining quantified the expression of inflammation-associated cytokines in skin tissue for the anti-inflammatory effects of ZIF-8@ Rutin. Treatment with ZIF-8@ RutinThe mouse tissue of (1) has the TNF-alpha content which is a typical proinflammatory factor obviously higher than that of a control group, and the Arg-1 content which is a typical anti-inflammatory factor obviously lower than that of the control group, and the excellent anti-inflammatory capability of the ZIF-8@ Rutin nano composite material in vivo is determined (figure 12).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A ZIF-8@ Rutin nano composite material with antibacterial and anti-inflammatory functions is characterized by being prepared from ZIF-8 nano particles and rutin.

2. The nanocomposite as claimed in claim 1, wherein the mass ratio of the ZIF-8 nanoparticles to rutin is 1.

3. The nanocomposite as claimed in claim 1, wherein the ZIF-8 nanoparticles have a particle size of 40 ± 3nm and the ZIF-8@ rutin nanocomposite has a particle size of 62 ± 3nm.

4. The nanocomposite as recited in claim 1, wherein the ZIF-8 nanoparticles have a dodecahedral morphology; the ZIF-8@ Rutin nano composite material is a secondary particle formed by agglomeration of primary particles.

5. A process for the preparation of a nanocomposite material according to any one of claims 1 to 4, comprising the steps of:

6. The preparation method according to claim 5, wherein the ZIF-8 nanoparticles are prepared as follows:

7. The preparation method according to claim 5, wherein the mass ratio of the ZIF-8 nanoparticles to the rutin is 1.

8. The method of claim 5, wherein the organic solvent is selected from methanol.

9. The method according to claim 5, wherein the heating is performed at a temperature of 50 ℃ to 55 ℃.

10. Use of a nanocomposite material according to any one of claims 1 to 4 in the manufacture of a medicament having antibacterial and anti-inflammatory effects and promoting wound healing.