CN116983328A

CN116983328A - Nanometer enzyme complex and application thereof

Info

Publication number: CN116983328A
Application number: CN202311247723.7A
Authority: CN
Inventors: 陈赣; 肖瑶; 张笑庸; 朱凯; 尤国兴; 周虹
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2023-09-26
Filing date: 2023-09-26
Publication date: 2023-11-03
Anticipated expiration: 2043-09-26
Also published as: CN116983328B

Abstract

The invention relates to the technical field of nano materials, in particular to a nano enzyme complex and application thereof. The nanoenzyme complex includes: prussian blue nanoenzyme; and silver ions and resveratrol coated on the surface of the Prussian blue nano enzyme. The nano enzyme complex can realize the effective treatment of sepsis by cooperating the functions of Prussian blue nano enzyme and a metal-polyphenol network to resist bacteria and inflammation, remove active oxygen, regulate immune homeostasis and relieve multi-organ injury, and can also be effectively applied to application scenes of eliminating inflammation, resisting bacteria and healing and repairing wound surfaces.

Description

Nanometer enzyme complex and application thereof

Technical Field

The invention relates to the technical field of nano materials, in particular to a nano enzyme complex and application thereof.

Background

Sepsis is a dysfunction of life-threatening organs caused by a deregulation of the host's response to infection. Despite the great advances in current therapies, sepsis remains a significant global challenge, with over 800 tens of thousands of deaths annually. The most advanced methods for treating severe sepsis and sepsis shock at present are mainly organ-supportive treatment, which requires intensive care of the patient, but has limited therapeutic effects. Therefore, finding an effective treatment for sepsis is an urgent need for human health.

Most clinical trials have been rarely successful due to the complexity of the pathogenic mechanisms of sepsis. First, the etiology of sepsis involves bacteria (gram negative and gram positive), viruses and fungal pathogens. When an infectious pathogen invades, the host immune system is activated, defending against the pathogen through a variety of molecular or cellular-based immune responses. The host immune response to sepsis is a complex process, with the initial excessive pro-inflammatory response lasting for days, leading to apoptosis of immune cells, and then progressing to a compensatory immunosuppressive phase, ultimately leading to immune dysfunction and death. Currently, antibiotics are the first-line drugs for clinical treatment of bacterial infections. However, due to the abuse of antibiotics and the long-term natural selection of bacteria, drug-resistant bacteria are continuously appeared, which constitutes a serious threat to the clinical application of the existing antibiotics. On the other hand, the immunotherapeutic concept for sepsis has also evolved from a single anti-inflammatory treatment to maintaining immune balance. Clinical trials of thymosin alpha 1 for the treatment of sepsis have recently been published. One randomized control study included 42 sepsis patients, and the results showed that patients in the ta 1 treated group had significantly lower mortality than the control group. At the same time, the mechanical ventilation time and Intensive Care Unit (ICU) hospital stay were shorter for the ta 1 treatment group. However, the use of immunomodulatory therapies is limited because of the variability in immune status of sepsis patients, and the timing of dosing is difficult to determine. At the same time, oxidative stress has been shown to play a major role in the progression of sepsis. However, clinically common antioxidants, such as vitamin C, have shown limited efficacy in large randomized clinical trials and reports suggest that the use of increased doses of vitamin C in sepsis patients may be detrimental.

Therefore, the search for effective methods of preventing secondary infections, restoring immune homeostasis, and alleviating multiple organ damage is of great importance in the treatment of sepsis.

Disclosure of Invention

The invention provides a nano enzyme complex, which comprises the following components:

prussian blue nanoenzyme; and silver ions and resveratrol coated on the surface of the Prussian blue nano enzyme.

The invention discovers that the metal-polyphenol network constructed by adopting silver ions and resveratrol can promote the function of Prussian blue nano-enzyme in scavenging active oxygen, and meanwhile, the Prussian blue nano-enzyme promotes the antibacterial and anti-inflammatory activities of the metal-polyphenol network, and the nano-enzyme compound constructed by the three can cooperatively play the roles of resisting bacteria, scavenging active oxygen and regulating immune balance.

Moreover, through mechanism research, the invention also discovers that the nano-enzyme complex can effectively inhibit lymphocyte apoptosis, and effectively improves the treatment effect on sepsis through the synergistic effect of the nano-enzyme complex and the antibacterial and active oxygen scavenging functions.

Preferably, the particle size of the nano enzyme complex is 120-180 nm.

More preferably, the particle size of the nano enzyme complex is 140-160 nm.

In some embodiments, the nanoenzyme complex has antibacterial activity.

In some embodiments, the nanoenzyme complex has superoxide dismutase catalytic activity.

In some embodiments, the silver ions are derived from silver nitrate.

Further, the present invention provides a method for preparing the nano-enzyme complex in any one of the above embodiments, comprising: and (3) dropwise adding a silver ion-containing solution and a resveratrol solution into the Prussian blue nano-enzyme solution.

Preferably, the concentration of the Prussian blue nano enzyme in the Prussian blue nano enzyme solution is 5-15 mg/mL.

Preferably, the concentration of silver ions in the silver ion-containing solution is 20-40 mM.

Preferably, the concentration of resveratrol in the resveratrol solution is 20-30 mM.

In some embodiments, the method of making further comprises: stirring for more than 5s after the dripping is finished, centrifugally cleaning at more than 8000rpm, and drying.

Preferably, the stirring is carried out for more than 10 seconds, and the centrifugal washing is carried out at more than 10000 rpm.

Further, the present invention provides a pharmaceutical product comprising the nanoenzyme complex of any one of the above embodiments, or the nanoenzyme complex produced by any one of the above production methods.

In some embodiments, the pharmaceutical product further comprises a pharmaceutically acceptable excipient.

In some embodiments, the excipients include, but are not limited to, fillers, excipients, lubricants, wetting agents, diluents.

In some embodiments, the formulation type of the drug product includes, but is not limited to, solid formulations (e.g., powders, granules, capsules, tablets, etc.), liquid formulations (e.g., oral liquids, etc.).

Preferably, the medicament is for the treatment of sepsis or for wound healing repair.

Further, the present invention provides a reagent or kit comprising the nanoenzyme complex of any one of the above embodiments, or the nanoenzyme complex produced by any one of the above production methods.

In specific embodiments, the agent or kit includes, but is not limited to, an agent for inhibiting bacterial growth or inhibiting bacterial activity, mimicking superoxide dismutase catalytic activity, scavenging active oxygen, or modulating immune homeostasis.

Further, the invention provides application of the nano-enzyme complex, or the nano-enzyme complex prepared by the preparation method, or medicines, reagents or kits in detecting or inhibiting bacterial growth or bacterial activity in the environment.

Further, the invention provides application of the nano-enzyme complex, or the nano-enzyme complex prepared by the preparation method, or a reagent or a kit in preparation of medicines; the medicine is used for treating sepsis, or eliminating inflammation, or antibacterial, or wound healing repair.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a nano enzyme complex, which can realize the effective treatment of sepsis by cooperating with the functions of Prussian blue nano enzyme and a metal-polyphenol network to resist bacteria and inflammation, remove active oxygen, regulate immune homeostasis and relieve the function of multiple organ injury, and can also be effectively applied to the application scenes of eliminating inflammation, resisting bacteria and healing and repairing wound surfaces.

Drawings

Fig. 1 is a TEM photograph of Prussian blue nanoenzyme.

FIG. 2 is a TEM photograph of the nanoenzyme complex.

FIG. 3 is a graph showing the zeta potential detection result of nanoparticles.

FIG. 4 is a graph of scanning electron microscope and energy dispersive X-ray characterization results.

Fig. 5 is a graph of the antibacterial results of the nanoenzyme complex and Prussian blue nanoenzyme 6 h.

FIG. 6 is a statistical graph of the catalytic activity of SOD at various concentrations of PB and ARPB.

Fig. 7 is a statistical plot of sepsis mice survival.

FIG. 8 is an immunofluorescence of spleen lymphocytes from sepsis mice.

Figure 9 is a statistical plot of the number of lymphocytes in the blood of sepsis mice.

Fig. 10 is a graph showing the results of promoting wound healing with the nano-enzyme complex and Prussian blue nano-enzyme.

In the figure, p value <0.05; * Represents p value <0.001; * P-value <0.0001.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The examples are not intended to identify the particular technology or conditions, and are either conventional or are carried out according to the technology or conditions described in the literature in this field or are carried out according to the product specifications. The reagents and instruments used, etc. are not identified to the manufacturer and are conventional products available for purchase by regular vendors.

Example 1

The embodiment provides a nano enzyme complex, which is prepared by the following steps:

1. preparation of Prussian blue nanoenzyme:

accurately weighing 3g of polyvinylpyrrolidone powder and 66mg of potassium ferricyanide solid by using an electronic analytical balance, sequentially dissolving in 40mL of 0.01M hydrochloric acid solution, and fully and uniformly mixing on a magnetic stirrer to obtain yellow clear solution; and (3) heating the obtained yellow clear solution in a muffle furnace at 80 ℃ for 20 hours, taking out the blue solution from the muffle furnace after cooling to normal temperature, centrifuging at 12000 rpm, washing for 3 times, drying to obtain blue Prussian blue nano enzyme (PB), dispersing PB nano particles in water, and placing in an ultrasonic oscillator for ultrasonic treatment for 3 minutes to ensure that the PB nano particles are uniformly dispersed. Then, a proper amount of PB solution is dripped on a copper mesh, and a Transmission Electron Microscope (TEM) is adopted for shooting, so that the PB is in a uniform cubic shape, the surface is smooth, the monodispersity is good, and the average size is 150 nm, which shows that the Prussian blue nano-enzyme is successfully prepared;

2. preparing a nano enzyme complex:

(1) Accurately weighing the Prussian blue nano enzyme (PB) prepared by using an electronic analytical balance, dissolving the Prussian blue nano enzyme (PB) in deionized water to prepare 9mg/mL PB solution, and placing the PB solution in an ultrasonic oscillator for ultrasonic treatment for 3min to uniformly disperse the PB solution;

(2) Accurately weighing 21.91mg of resveratrol (Res) by an electronic analytical balance, and dissolving in 4mL of deionized water to prepare 24mM resveratrol water solution;

(3) Accurately weighing 5.10mg of silver nitrate by using an electronic analytical balance, and dissolving the silver nitrate in 1mL of deionized water to prepare 30mM silver nitrate solution;

(4) And (3) simultaneously and slowly dripping 4mL of resveratrol aqueous solution and 1mL of silver nitrate solution into the PB solution, stirring for 10s after dripping, centrifuging at 10000rpm, washing for 3 times, drying to obtain a blue nano enzyme compound (Ag-Res-PB, ARPB for short), and observing by TEM (transverse magnetic) shooting, wherein the result is shown in figure 2, and the result shows that Prussian blue nano enzyme shows that a thin shell is coated, so that silver ions and resveratrol are successfully coated. Further, it was found by examination of the zeta potential that PB has a zeta potential of-10.70.+ -. 0.10 mV and ARPB has a zeta potential of-24.13.+ -. 0.57 mV, and successful encapsulation of silver ions and resveratrol was also confirmed (FIG. 3). Fe, O, C and Ag were found to coexist with ARPB by scanning electron microscopy and energy dispersive X-ray characterization (FIG. 4).

Example 2

This example tests the antimicrobial activity of the nanoenzyme complex prepared in example 1. The method comprises the following specific steps:

gram-positive staphylococcus aureus and gram-negative escherichia coli are selected as model bacteria. The antibacterial activity of PB and ARPB was evaluated by agar plate counting. After incubation of ARPB and PB at 10. Mu.g/mL concentration for 6h at 37℃respectively, the bacteriostatic effect was determined, with PBS as control.

The results are shown in FIG. 5, where the PB group had a similar number of bacterial colonies to the PBS group, while the ARPB group had no bacterial colonies, indicating that the ARPB could kill or resist bacterial adhesion.

Example 3

The catalytic activity of superoxide dismutase (SOD) is simulated to reflect the antioxidant capacity of the nanoparticles. SOD acts as a specific enzyme that degrades superoxide, plays an important role in active oxygen balance, and acts as an antioxidant to protect cellular components from oxidative damage by superoxide. This example uses the hydroxylamine method to evaluate the catalytic activity of the nanoparticle prepared in example 1 to mimic SOD.

The results showed that ARPB at 0.03mg/mL, 0.3mg/mL and 3mg/mL exhibited stronger catalytic activity for the simulated SOD than PB at the same concentration (FIG. 6), demonstrating that silver ions and resveratrol were able to promote catalytic activity for the simulated SOD for PB.

Example 4

To further explore the therapeutic potential of ARPB for sepsis, this example assessed the effect of ARPB treatment on survival of lethal sepsis (CLP) mice. The method comprises the following specific steps:

wild type BALB/c mice (SPF grade, male, 6-8 weeks, 20-25 g) were placed in the animal house for 3 days of adaptive rearing, and no water was allowed to enter for 10 h before the experiment, and a cecal ligation perforated sepsis model mouse (CLP mouse) was constructed, specifically: randomly dividing the mice fasted in advance into a sham group and a CLP group, wherein 11 mice in each group are anesthetized by intraperitoneal injection by sucking 5% chloral hydrate (10 mL/kg) by a 1mL injector; after anesthesia, fixing the supine position of the mouse on an operation board, disinfecting the abdomen of the mouse by using iodophor, making a longitudinal incision along the white line of the abdomen at a position 1cm below the xiphoid process by using ophthalmic scissors, separating the cecum and ligating at a position about 1cm away from the tail end of the cecum, then puncturing the tail end of the cecum once by using a 22G puncture needle head at the position avoiding blood vessels, extruding the cecum to overflow the excrement, then returning the cecum to the original position, suturing the incision of the abdomen, sterilizing by using iodophor, and gently softening the abdomen; finally, 1mL of 0.9% physiological saline is injected into the subcutaneous part of the cervical of the mouse by a 5 mL injector, and the postoperative mouse is warmed. NS group mice were intraperitoneally injected with 0.2mL of physiological saline. PB group mice were intraperitoneally injected with 9mg/mL of PB solution 0.2mL and ARPB group mice were intraperitoneally injected with 9mg/mL of ARPB solution 0.2mL.

As shown in fig. 7, mice died within 24 hours of CLP group, and single administration of ARPB significantly improved CLP mice survival from 18.2% to 72.7% compared to PB group.

Example 5

This example further explores the effect of ARPB on CLP mice spleen CD4 and CD8 to demonstrate the effect of ARPB on CLP mouse immune function. The specific experimental steps are as follows:

dissecting the CLP mice in the example 4, fixing spleen tissues in 4% paraformaldehyde, taking out the fixed spleen tissues, embedding the fixed spleen tissues into paraffin sections, placing the paraffin sections into EDTA antigen retrieval buffer (pH 8.0) and performing antigen retrieval in a microwave oven, sealing the sections for 30min by adopting serum, adding a CD4 primary antibody for incubation overnight, adopting an HRP-labeled secondary antibody for incubation at room temperature for 50min, washing the sections by adopting PBS, adding TSA for light-shielding incubation for 10min, adding the CD8 primary antibody after antigen retrieval, adopting the HRP-labeled secondary antibody for incubation for 50min, counterstaining cell nuclei for 10min by adopting DAPI, putting the sections into PBS for washing after spin-drying, adding an autofluorescence quenching agent for incubation for 5min, washing the sections, and scanning the sections by adopting a fluorescence source.

The results are shown in fig. 8 (grey part is fluorescence), CK is a normal mouse group, and the results indicate that ARPB can significantly increase the number of T lymphocytes in spleen compared to PB.

In addition, the number of lymphocytes in the blood of CLP mice was also detected in this example, and as shown in fig. 9, ARPB can significantly increase the number of T lymphocytes in the spleen compared to PB, indicating that ARPB can effectively improve immune organ damage caused by sepsis.

Example 6

The embodiment detects the wound repair function of ARPB, and specifically comprises the following steps:

(1) Material preparation

Adding 2% HA (hyaluronic acid) and 3% SA (sodium alginate) into double distilled water, and uniformly mixing to obtain blank water gel; adding 2% HA and 3% SA into 10mg/mL PB solution, and uniformly mixing to obtain PB gel; adding 2% HA and 3% SA into 10mg/mL ARPB solution, and uniformly mixing to obtain ARPB gel;

(2) Mice were anesthetized with isofluraneShaving back hair, making a wound with a diameter of 10 mm on skin with sterile ophthalmic scissors, and suspending methicillin-resistant Staphylococcus aureus (50 μl,1×10) ⁷ CFU/mL) is gradually applied to the wound and allowed to dry naturally. The random groups were 4, 5 per group: (1) control CK (no drug administration), (2) blank hydrogel CG, (3) PB hydrogel, and (4) ARPB hydrogel; the same dose of drug was applied to the wound surface of the mice at the same time on days 1, 3, 5, 7, 9, respectively, and the wound was photographed.

The results are shown in figure 10, and ARPB can effectively promote wound repair of mice.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanoenzyme complex comprising:

2. The nano-enzyme complex according to claim 1, wherein the particle size of the nano-enzyme complex is 120-180 nm.

3. The nanoenzyme complex of claim 1 or 2, wherein said nanoenzyme complex has antibacterial activity.

4. The nano-enzyme complex according to claim 1 or 2, wherein the nano-enzyme complex has superoxide dismutase catalytic activity.

5. The nanoenzyme complex of claim 1 or 2, wherein the silver ions are derived from silver nitrate.

6. The method for preparing the nano-enzyme complex according to any one of claims 1 to 5, comprising the steps of: and (3) dropwise adding a silver ion-containing solution and a resveratrol solution into the Prussian blue nano-enzyme solution.

7. A pharmaceutical product comprising the nanoenzyme complex according to any one of claims 1 to 5 or the nanoenzyme complex produced by the production method according to claim 6.

8. A reagent or kit comprising the nanoenzyme complex according to any one of claims 1 to 5 or the nanoenzyme complex produced by the production method according to claim 6.

9. Use of the nanoenzyme complex of any one of claims 1-5, or the nanoenzyme complex prepared by the preparation method of claim 6, or the pharmaceutical product of claim 7, or the reagent or kit of claim 8, for detecting or inhibiting bacterial growth or bacterial activity in an environment.

10. Use of the nanoenzyme complex of any one of claims 1-5, or the nanoenzyme complex prepared by the preparation method of claim 6, or the reagent or kit of claim 8 in the preparation of a medicament;

the medicine is used for treating sepsis, or eliminating inflammation, or antibacterial, or wound healing repair.