CN114681482B

CN114681482B - Nanometer enzyme and preparation method and application thereof

Info

Publication number: CN114681482B
Application number: CN202111004876.XA
Authority: CN
Inventors: 黄鹏; 涂天慧; 张东阳; 林静
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2021-08-30
Filing date: 2021-08-30
Publication date: 2023-05-05
Anticipated expiration: 2041-08-30
Also published as: CN114681482A

Abstract

The invention discloses a nano-enzyme, a preparation method and application thereof, wherein the nano-enzyme comprises rhodium nano-particles and serine combined on the surfaces of the rhodium nano-particles. The nano-enzyme (Rh-Ser) provided by the invention has an ultra-small size which is lower than the threshold value of tubular filtration; meanwhile, serine has the capability of targeting kidney injury molecule-1 (KIM-1) expressed by a damaged renal tubule, so that the rhodium nanoparticle has a certain kidney targeting capability due to serine modification, can be effectively enriched in the kidney of a damaged mouse, can remove a large amount of active oxygen or active nitrogen in the renal tubule to relieve and treat acute kidney injury induced by glycerol, and has an excellent anti-inflammatory capability. In addition, these Rh-Ser have good therapeutic effects, and have excellent biocompatibility and biosafety.

Description

Nanometer enzyme and preparation method and application thereof

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a nano-enzyme and a preparation method and application thereof.

Background

Acute kidney injury is an important health problem in humans, with high morbidity and mortality, estimated to die of the acute kidney injury problem in 170 tens of thousands of people worldwide each year. Currently, adjuvant therapy and kidney transplantation are the most common treatments for acute kidney injury. Recent studies have shown that the pathogenesis of acute kidney injury is associated with intracellular excess reactive oxygen and reactive nitrogen species. Heretofore, some small molecule drugs, such as: amifostine and acetylcysteine have been shown to act as antioxidants, eliminating reactive oxygen species, and thus alleviating acute kidney injury. However, the existing small molecule drugs have no targeting property, so that the utilization rate is low, large toxic and side effects are easy to cause, the curative effect is limited, and the clinical application of the small molecule drugs is hindered.

Accordingly, the prior art is still in need of improvement and development.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide nano-enzyme and a preparation method and application thereof, and aims to solve the technical problems that the existing small-molecule drug is low in utilization rate and large in side effect, and is difficult to treat acute kidney injury.

In a first aspect of the present invention, there is provided a nanoenzyme comprising rhodium nanoparticles and serine bound to the surface of the rhodium nanoparticles. The rhodium nano-enzyme has the capability of targeting damaged kidneys due to the existence of the surface ligand serine, and has good water solubility and biological safety, is not easy to act on proteins in serum, and is beneficial to the circulation of nano-particles in blood.

Optionally, the mass ratio of the rhodium nanoparticle to the serine is 1 (1-4).

Optionally, the nanoenzyme is a spherical particle with a diameter of less than 5.5 nm.

In a second aspect of the present invention, there is provided a method for preparing a nanoenzyme as described above, comprising the steps of: rhodium chloride trihydrate and methoxy polyethylene glycol mercapto are mixed in water, sodium borohydride is added after uniform ultrasonic dispersion, and rhodium nanoparticle solution is prepared by reaction; and (3) after ultrafiltration washing is carried out on the rhodium nanoparticle solution, serine is added and stirred, so that serine is combined on the surface of the rhodium nanoparticle, and the nano enzyme is prepared.

Optionally, the mass ratio of rhodium chloride trihydrate to methoxy polyethylene glycol mercapto is 3 (1-10).

Alternatively, the reaction time is 5-30 minutes, and the reaction temperature is room temperature.

Optionally, the mass ratio of rhodium chloride trihydrate to serine is 1 (1-4).

In a third aspect, the invention provides an application of the nano-enzyme in preparing a drug for targeted treatment of acute kidney injury.

Optionally, the medicament is in the form of capsule, tablet, oral preparation, injection, suppository, spray or ointment.

The beneficial effects are that: the nano-enzyme provided by the invention comprises rhodium nano-particles and serine combined on the surfaces of the rhodium nano-particles. The nano-enzyme (Rh-Ser) has ultra-small size which is lower than the filtration threshold of the renal tubule, and serine has the capability of targeting the renal injury molecule-1 (KIM-1) expressed by the damaged renal tubule, so that the modification of serine can enable rhodium nano-particles to have certain renal targeting capability, so that the rhodium nano-particles can be effectively enriched in the kidney of a damaged mouse, and a large amount of active oxygen or active nitrogen in the renal tubule is removed to relieve and treat the acute renal injury induced by glycerol. In addition, these Rh-Ser have good therapeutic effects, and have excellent biocompatibility and biosafety.

Drawings

FIG. 1 is a synthetic scheme of Rh-Ser in an embodiment of the invention;

FIG. 2 is an Atomic Force Microscope (AFM) view of Rh-Ser and a height view of the AFM in an embodiment of the invention;

FIG. 3 is a high resolution transmission electron microscope (HR-TEM) image of Rh-Ser in an embodiment of the present invention;

FIG. 4 is an X-ray diffraction (XRD) pattern of Rh-Ser in an embodiment of the invention;

FIG. 5 is a graph showing the scavenging rate of Rh-Ser hydroxyl radicals in an embodiment of the invention;

FIG. 6 is a graph showing the superoxide anion clearance of Rh-Ser in an embodiment of the invention;

FIG. 7 is a graph of the free radical scavenging by the 2,2' -diaza-bis (3-ethylbenzothiazole-6-sulfonic acid) diammonium salt (ABTS) of Rh-Ser in an embodiment of the invention;

FIG. 8 is a graph showing the nitrogen radical scavenging rate of Rh-Ser in an embodiment of the invention, 2-biphenyl-1-picrylhydrazyl (DPPH);

FIG. 9 is a graph showing Rh-Ser treated tubular cells (293T) and human tubular epithelial cells (HK-2) viability in an embodiment of the invention;

FIG. 10 shows the Rh-Ser and H concentrations in an embodiment of the invention ₂ O ₂ Viability graph after incubation of stimulated human tubular epithelial cells (HK-2);

FIG. 11 is a graph of photoacoustic imaging of the kidney and a semi-quantitative graph of photoacoustic results of mice under different treatments for Rh-Ser versus rhodium nanoenzymes without modification and threonine modification in a specific example of the present invention;

FIG. 12 is a graph showing Rh-Ser levels of interleukin-6 (IL-6) and tumor necrosis factor (TNF-. Alpha.) in mice in different treatment groups according to an embodiment of the present invention;

FIG. 13 is a graph showing the Blood Urea Nitrogen (BUN) content of Rh-Ser in the serum of mice of different treatment groups according to an embodiment of the present invention;

FIG. 14 is a graph showing the serum Creatinine (CREA) content of Rh-Ser in mice of different treatment groups according to an embodiment of the present invention;

FIG. 15 is a graph showing the change of body weight with time of mice with acute renal failure injected with phosphate buffer alone in the embodiment of the present invention.

FIG. 16 is a graph showing the weight of Rh-Ser-injected acute renal failure mice over time in an embodiment of the present invention.

Detailed Description

The invention provides a nano-enzyme, a preparation method and application thereof, and the invention is further described in detail below in order to make the purposes, technical schemes and effects of the invention clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The inventor researches find that compared with the traditional protease, the nano-enzyme has the obvious advantages of low cost, adjustable catalytic property, large-scale preparation and the like. Meanwhile, the research discovers that rhodium nano-enzyme has broad-spectrum active oxygen and active nitrogen scavenging capability. Furthermore, nanomaterials have unique physicochemical properties that make them useful as contrast agents for clinical or preclinical imaging modalities. More importantly, the ultra-small nanoparticles can be metabolized by the kidneys, which provides the possibility for the treatment of acute kidney injury. Research proves that serine has the capability of targeting kidney injury molecule-1 (KIM-1) expressed by damaged renal tubules, so that the serine is modified to enable the material to have certain damaged kidney targeting capability.

Based on this, the embodiment of the invention provides a nano-enzyme, which comprises rhodium nano-particles and serine combined on the surfaces of the rhodium nano-particles. The nanoenzyme in this embodiment may be abbreviated as Rh-Ser.

The Rh-Ser provided in this example has an ultra-small size, below the tubular filtration threshold; meanwhile, serine has the capability of targeting kidney injury molecule-1 (KIM-1) expressed by a damaged renal tubule, so that the rhodium nanoparticle has a certain kidney targeting capability by serine modification, can be effectively enriched in the kidney of a damaged mouse, can remove a large amount of active oxygen or active nitrogen in the renal tubule to relieve and treat acute kidney injury induced by glycerol, and has excellent anti-inflammatory capability. In addition, these Rh-Ser have good therapeutic effects, and have excellent biocompatibility and biosafety.

In the implementation, the serine is modified to ensure that the rhodium nano-enzyme has certain impaired kidney targeting capability, and the rhodium nano-enzyme has good water solubility and biological safety, is not easy to react with proteins in serum, and is beneficial to the circulation of rhodium nano-particles in blood.

In one embodiment, the mass ratio of the rhodium nanoparticle to the serine is 1: (1-4).

In one embodiment, the Rh-Ser is a spherical particle with a diameter of less than 5.5nm, below the renal tubular filtration threshold. The serine can be effectively enriched in the kidney of a damaged mouse due to modification of serine, and the ultra-small nano particles are beneficial to metabolism through the kidney.

The embodiment of the invention provides a preparation method of Rh-Ser, which comprises the following steps: rhodium chloride trihydrate and methoxy polyethylene glycol mercapto are mixed in water, sodium borohydride is added after uniform ultrasonic dispersion, and rhodium nanoparticle solution is prepared by reaction; and (3) after ultrafiltration washing is carried out on the rhodium nanoparticle solution, serine is added and stirred, so that serine is combined on the surface of the rhodium nanoparticle, and the nano enzyme is prepared.

In some specific embodiments, rhodium chloride trihydrate and methoxy polyethylene glycol mercapto are mixed in water according to the mass ratio of 3 (1-10), uniformly dispersed by ultrasonic and stirred for 10-15 minutes, then sodium borohydride (2 mg/mL) which is prepared at present is added dropwise, and the mixture is reacted for 5-30 minutes at room temperature (18-35 ℃); ultrafiltration washing the obtained solution, adding serine and stirring overnight; and ultrafiltering and washing the obtained solution to obtain the Rh-Ser.

In some embodiments, the application of Rh-Ser in preparing the drug for targeted treatment of acute kidney injury is also provided.

Preferably, the medicament is in the form of a capsule, tablet, oral preparation, injection, suppository, spray or ointment.

The technical scheme of the invention is further described by specific examples.

Example 1: synthesis of Rh-Ser

Rh-Ser synthesis: 60mg RhCl ₃ ·3H ₂ O was dissolved in 50mL of water, followed by addition of 40mg of methoxypolyethylene glycol mercapto group to the solution and vigorous stirring. After 0.5h, 5mL NaBH was quickly added dropwise to the mixture ₄ Aqueous solution (2 mg/mL). After 10 minutes of reaction, the above solution was purified by ultrafiltration centrifugation. The sample was then freeze-dried to give a solid product. The dried solid product was then dispersed in water (2 mg/mL,5 mL) and mixed with L-serine and stirred overnight to give Rh-Ser. The free L-serine was removed by ultrafiltration centrifugation and the purified solution was lyophilized for ready use. The threonine modified rhodium nanoenzyme replaces serine with threonine, and the obtained sample is called Rh-Thr.

FIG. 1 is a scheme for the synthesis of Rh-Ser, wherein RhCl ₃ ·3H ₂ O represents rhodium chloride trihydrate and L-Ser represents serine. The Rh-Ser has a certain ability to target the damaged tubular.

FIG. 2 is an AFM image of the synthesized Rh-Ser; FIG. 3 is an HR-TEM image of synthetic Rh-Ser; FIGS. 2 and 3 show that Rh-Ser has an ultra-small size. FIG. 4 is an XRD pattern for Rh-Ser showing that rhodium nanoenzyme is the standard tetrahedral structure.

Example 2: rh-Ser has the ability to scavenge a variety of active oxygen/nitrogen.

The efficiency of scavenging hydroxyl radicals at different concentrations of Rh-Ser (0-100. Mu.g/mL) was determined by the hydroxyl radical antioxidant capacity (HORAC) kit (Cell Biolabs, USA). The test was performed according to the protocol provided by the manufacturer.

As shown in FIG. 5, rh-Ser is capable of efficiently scavenging hydroxyl radicals and has concentration-dependent properties.

The efficacy of different concentrations of Rh-Ser (0-5. Mu.g/mL) in scavenging superoxide anions was determined by the superoxide dismutase (SOD) detection kit (Sigma-Aldrich, USA). The test was performed according to the protocol provided by the manufacturer.

As shown in FIG. 6, rh-Ser is capable of effectively scavenging superoxide anions and has concentration-dependent properties.

Test of Rh-Ser scavenging 2,2' -diazabis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) free radical

The radical scavenging capacity of Rh-Ser was determined by the ABTS radical cation decolorization method. ABTS (7 mM) is dissolved in water and reacted with 2.45mM potassium persulfate for half an hour to produce ABTS radical cations (. ABTS) ⁺ ). Then the pure ABTS was determined at 734nm ⁺ Solutions (AB) and different concentrations (0-10. Mu.g/mL) of Rh-Ser and. ABTS ⁺ Absorbance value of the mixed solution. The calculation formula of the ABTS cleaning efficiency is [ (AB-AP)/AB]*100. All measurements were performed in triplicate.

As shown in FIG. 7, rh-Ser is capable of efficiently scavenging ABTS free radicals and has concentration-dependent properties.

Rh-Ser scavenging nitrogen radical 2, 2-biphenyl-1-picrylhydrazyl (DPPH)

The activity of Rh-Ser on active nitrogen scavenging was evaluated using DPPH. After mixing Rh-Ser (0-100. Mu.g/mL) at various concentrations with 100. Mu.M DPPH for 2 hours, the absorbance (A) of the mixture at 550nm was measured _Sample ) And absorbance of DPPH at 550nm (A _{For a pair of} )。DThe calculation formula of the PPH cleaning efficiency is [ (A) _{For a pair of} -A _Sample )/A _{For a pair of} ]*100. All measurements were performed in triplicate.

As shown in FIG. 8, rh-Ser is capable of effectively scavenging active nitrogen DPPH and has a concentration-dependent characteristic.

Example 3: rh-Ser cytotoxicity and the effect of Rh-Ser on 293T kidney embryonic and human tubular epithelial cells (HK-2) viability was evaluated by scavenging various reactive oxygen/reactive nitrogen protected kidney cells using standard MTT methods.

293T cells or HK-2 cells at 1X 10 per well ⁴ Density inoculation into 96 well plates and exposure to 37 degrees celsius, 5% co ₂ Incubate for 12h under conditions. Next, old medium in the 96-well plate was aspirated, and medium solutions containing Rh-Ser at different concentrations were added, respectively. After further culturing for 20 or 44 hours, the old medium in the 96-well plate was aspirated, and 100. Mu.L of MTT medium solution (0.8 mg/mL) was added to each well, and culturing was continued for 4 hours. The residual medium in the 96-well plate was aspirated, 150. Mu.L of DMSO solution was added to each well, and after gentle shaking, the OD value (detection wavelength: 570 nm) of each well was measured on a Synergy H1-type microplate reader, and the cell viability was calculated using the following formula. Cell viability (%) = (OD of sample) ₅₇₀ Value/blank OD ₅₇₀ Value) x 100%.

As shown in FIG. 9, the cell viability of the synthesized Rh-Ser on 293T kidney embryonic cells and human tubular epithelial cells (HK-2) remained at greater than 80% when the concentration reached a maximum use concentration of 200. Mu.g/mL. The Rh-Ser of the invention is shown to have lower cytotoxicity.

As shown in FIG. 10, after 293T cells were treated with Rh-Ser (0-200. Mu.g/mL) 4 hours in advance, they were cultured in a medium containing 0.5mM hydrogen peroxide for 20 hours and then the cell viability was measured by the MTT method described above. Cell viability increased with increasing Rh-Ser concentration, indicating Rh-Ser vs. H ₂ O ₂ Human tubular epithelial cells (HK-2) under stimulation have a concentration-dependent protective effect.

Example 4: rh-Ser kidney targeting, where all experimental procedures of photoacoustic imaging were in accordance with animal use and care regimen passed by the clinical center animal health and use Committee. Female athymic mice (six weeks, 20-25 g) were subjected to intramuscular injection of 8mL/kg of 50% glycerol solution into the hind legs of the mice to establish a mouse acute renal failure model (RM-AKI). After 2 hours, the tail vein was injected with Rh-Ser or unmodified rhodium nanoparticles Rh-NPs, threonine modified rhodium nanoparticles Rh-Thr.

The kidneys were taken out at different time points, and imaged using a photoacoustic imager. As shown in fig. 11, the photoacoustic signal of the Rh-Ser group injected mice was significantly enhanced, and the signal intensity reached the peak at 4 hours, which was much higher than that of the other groups, indicating that Rh-Ser had excellent impaired kidney targeting ability.

Example 5: rh-Ser treatment of acute renal injury and evaluation of biosafety

All experimental procedures were in accordance with animal use and care regimens passed by the institutional animal care and use committee of clinical centers. Female athymic mice (six weeks, 20-25 g), mice were developed in a model of acute renal failure by intramuscular injection of 8mL/kg of 50% glycerol solution into the hind legs of the mice. After 2 hours, the small molecule drug acetylcysteine or Rh-Ser was injected into the tail vein.

Mice were randomly divided into 5 groups: (1) healthy mice were injected with phosphate buffer; (2) healthy mice were injected with Rh-Ser; (3) Glycerol-induced acute renal failure mice were injected with phosphate buffer; (4) Glycerol-induced acute renal failure mice were injected with Rh-Ser equivalent of acetylcysteine; (5) Glycerol-induced acute renal failure mice were injected with Rh-Ser. Wherein the volume of the phosphate buffer used for injection is 100 mu L, the injection dosage of Rh-Ser is 5mg/kg, and the injection dosage of acetylcysteine is 5mg/kg. Healthy mice and glycerol-induced acute renal failure mice were euthanized after 24 hours, the mice were blood centrifuged to obtain serum, and the kidneys of the mice were collected.

The kidneys of the mice were weighed and homogenized in 4mL of phosphate buffer, centrifuged at 5000rpm for 3 minutes, and the supernatants were taken to determine the levels of the pro-inflammatory factors tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in the kidney homogenates of the mice according to the instructions using IL-6 and TNF-alpha (Abbkine Scientific Company, USA) ELISA kit. As shown in FIG. 12, the levels of IL-6 and TNF-. Alpha.in Rh-Ser treated acute renal failure mice were reduced to normal, indicating that Rh-Ser had excellent anti-inflammatory therapeutic effects.

As shown in FIGS. 13-14, creatinine and blood urea nitrogen content were measured. The creatinine and blood urea nitrogen content of Rh-Ser injected by healthy mice did not change significantly. Whereas the Rh-Ser injected acute renal failure mice had significantly lower creatinine and blood urea nitrogen levels than mice injected with phosphate buffer alone and approached the levels of healthy mice. While the same dosage of acetylcysteine does not effectively reduce both indicators. This shows that Rh-Ser can effectively relieve and treat acute renal failure, and has better therapeutic effect than the small molecular medicine acetylcysteine used clinically.

In addition, mice with acute renal failure were injected with phosphate buffer and Rh-Ser, and weight changes were recorded over fifteen days. As shown in fig. 15 (phosphate buffer group) and fig. 16 (Rh-Ser group), rh-Ser-injected mice gradually recovered after weight reduction compared to the control group, indicating that Rh-Ser has a good therapeutic effect.

In conclusion, the Rh-Ser can be used for preparing a large amount of ultra-small nano particles through a simple synthesis method, can effectively remove various active oxygen/active nitrogen species, and has broad-spectrum active oxygen/active nitrogen removing capability. The toxicity and side effects on 293T kidney cells and human tubular epithelial cells (HK-2) are low, and the cell survival rate reaches more than 80% after the cells are co-cultured for 24 hours; while they can protect cells from hydrogen peroxide by scavenging excess reactive oxygen/nitrogen in the cells. Rh-Ser has the ability to target the kidney due to the modification of serine, and the excellent kidney targeting ability of Rh-Ser is confirmed by means of a photoacoustic imager. In addition, rh-Ser showed good therapeutic effects and excellent anti-inflammatory ability in glycerol-induced acute renal failure mice. More importantly, rh-Ser has good biocompatibility and biosafety.

It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.

Claims

1. A nanoenzyme comprising rhodium nanoparticles and serine bound to the surface of the rhodium nanoparticles;

the mass ratio of the rhodium nano-particles to the serine is 1:1-4;

the nano-enzyme is spherical particles with the diameter smaller than 5.5 nm.

2. A method of preparing the nanoenzyme of claim 1, comprising the steps of:

rhodium chloride trihydrate and methoxy polyethylene glycol mercapto are mixed in water, sodium borohydride is added after uniform ultrasonic dispersion, and rhodium nanoparticle solution is prepared by reaction;

and (3) after ultrafiltration washing is carried out on the rhodium nanoparticle solution, serine is added and stirred, so that serine is combined on the surface of the rhodium nanoparticle, and the nano enzyme is prepared.

3. The method for preparing nano-enzyme according to claim 2, wherein the mass ratio of rhodium chloride trihydrate to methoxy polyethylene glycol mercapto is 3:1-10.

4. The method for preparing nano-enzyme according to claim 2, wherein in the step of preparing rhodium nano-particle solution by reaction, the reaction time is 5-30min.

5. Use of a nanoenzyme according to claim 1 for the preparation of a medicament for targeted treatment of acute kidney injury.

6. The use of nanoenzyme according to claim 5, wherein the pharmaceutical dosage form is an oral preparation, injection, suppository, spray or ointment.