CN114681482A

CN114681482A - Nano enzyme and preparation method and application thereof

Info

Publication number: CN114681482A
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: 2022-07-01
Anticipated expiration: 2041-08-30
Also published as: CN114681482B

Abstract

The invention discloses a nano enzyme and 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 renal tubule filtration threshold; meanwhile, serine has the ability of targeting the kidney injury molecule-1 (KIM-1) expressed by the damaged renal tubules, so that modification of serine can enable the rhodium nanoparticles to have certain kidney targeting ability, the rhodium nanoparticles can be effectively enriched in the kidney of the damaged mouse, a large amount of active oxygen or active nitrogen in the renal tubules can be eliminated to relieve and treat acute renal injury induced by glycerol, and the rhodium nanoparticles have excellent anti-inflammatory ability. In addition, the Rh-Ser has good treatment effect, and simultaneously has excellent biocompatibility and biological safety.

Description

Nano 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 a significant health problem in humans with high morbidity and mortality rates, and it is estimated that 170 million people die of acute kidney injury every year worldwide. Currently, adjuvant therapy and kidney transplantation are the most common treatment of acute kidney injury. Recent studies have shown that the pathogenesis of acute kidney injury is associated with an excess of reactive oxygen and reactive nitrogen species within the cell. Previously, some small molecule drugs, such as: amifostine and acetylcysteine have been shown to act as antioxidants to eliminate reactive oxygen species, thereby alleviating acute kidney injury. However, the existing small molecule drugs have no targeting property, so 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 drugs is hindered.

Accordingly, the prior art is yet to be improved and developed.

Disclosure of Invention

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

In a first aspect of the invention, a nanoenzyme is provided, which comprises rhodium nanoparticles and serine bound on the surfaces of the rhodium nanoparticles. The existence of the surface ligand serine enables the rhodium nanoenzyme to have the capability of targeting the damaged kidney, and the rhodium nanoenzyme has good water solubility and biological safety, is not easy to generate the effect of protein in serum, and is beneficial to the circulation of nanoparticles in blood.

Optionally, the mass ratio of the rhodium nanoparticles 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 the nanoenzyme, comprising the steps of: mixing rhodium chloride trihydrate and methoxypolyethylene glycol mercapto in water, adding sodium borohydride after uniform ultrasonic dispersion, and reacting to obtain a rhodium nanoparticle solution; and (3) carrying out ultrafiltration washing on the rhodium nanoparticle solution, then adding serine, and stirring to combine the serine on the surface of the rhodium nanoparticle, thus obtaining the nano enzyme.

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

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

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

In a third aspect of the invention, an application of the nanoenzyme in preparation of a medicine for targeted therapy of acute kidney injury is provided.

Optionally, the medicament is in the form of capsules, tablets, oral preparations, injections, suppositories, sprays or ointments.

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

Drawings

FIG. 1 is a synthesis scheme of Rh-Ser in a specific example of the present 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 present invention;

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

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

FIG. 5 is a graph showing the hydroxyl radical scavenging rate of Rh-Ser in a specific example of the present invention;

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

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

FIG. 8 is a graph of Rh-Ser 2, 2-biphenyl-1-picrylhydrazino (DPPH) nitrogen radical scavenging efficiency for specific examples of the present invention;

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

FIG. 10 shows different concentrations of Rh-Ser and H in an embodiment of the present invention₂O₂Survival plots after incubation of stimulated human tubular epithelial cells (HK-2);

FIG. 11 is a graph of renal photoacoustic imaging and semi-quantitative photoacoustic results from Rh-Ser versus unmodified and threonine modified rhodium nanoenzymes in different treatments for mice in a specific example of the invention;

FIG. 12 is a graph of the levels of IL-6 (IL-6) and TNF (TNF- α) levels of Rh-Ser in the kidney of mice from different treatment groups in a particular example of the invention;

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

FIG. 14 is a graph of serum Creatinine (CREA) content of Rh-Ser in the serum of mice of different treatment groups in a specific example of the present invention;

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

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

Detailed Description

The invention provides a nano enzyme and a preparation method and application thereof, and the invention is further described in detail below in order to make the purpose, technical scheme and effect of the invention clearer and more clear. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The inventor finds 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, research finds that the rhodium nanoenzyme has broad-spectrum active oxygen and active nitrogen scavenging capacity. In addition, nanomaterials have unique physicochemical properties that make them useful as contrast agents for clinical or pre-clinical imaging modalities. More importantly, the ultra-small nanoparticles can be metabolized by the kidney, which provides the possibility for the treatment of acute kidney injury. Studies prove that serine has the capacity of targeting kidney injury molecule-1 (KIM-1) expressed by damaged renal tubules, so that modification of serine can enable the material to have certain damaged kidney targeting capacity.

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

Rh-Ser provided in this example is of ultra-small size, below the tubular filtration threshold; meanwhile, serine has the ability of targeting the kidney injury molecule-1 (KIM-1) expressed by the damaged renal tubules, so that modification of serine can enable the rhodium nanoparticles to have certain kidney targeting ability, the rhodium nanoparticles can be effectively enriched in the kidneys of damaged mice, a large amount of active oxygen or active nitrogen in the renal tubules can be removed to relieve and treat acute renal injury induced by glycerol, and the serine has excellent anti-inflammatory ability. In addition, the Rh-Ser has good treatment effect, and simultaneously has excellent biocompatibility and biological safety.

In the implementation, the modification of serine enables the rhodium nano-enzyme to have certain damaged kidney targeting capability, and the rhodium nano-enzyme has good water solubility and biological safety, is not easy to react with serum endoproteins, and is beneficial to the circulation of rhodium nano-particles in blood.

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

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

The embodiment of the invention provides a preparation method of Rh-Ser, which comprises the following steps: mixing rhodium chloride trihydrate and methoxypolyethylene glycol mercapto in water, adding sodium borohydride after uniform ultrasonic dispersion, and reacting to obtain a rhodium nanoparticle solution; and (3) carrying out ultrafiltration washing on the rhodium nanoparticle solution, then adding serine, and stirring to combine the serine on the surface of the rhodium nanoparticle, thus obtaining the nano enzyme.

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

In some embodiments, the invention further provides application of the Rh-Ser in preparation of a medicine for targeted treatment of acute kidney injury.

Preferably, the medicament is in the form of capsules, tablets, oral preparations, injections, suppositories, sprays or ointments.

The technical solution of the present invention is further illustrated by the following specific examples.

Example 1: synthesis of Rh-Ser

Rh-Ser synthesis: 60mg of RhCl₃·3H₂O was dissolved in 50mL of water, and then 40mg of methoxypolyethylene glycol mercapto group was added to the solution, and vigorously stirred. After 0.5h, 5mL of NaBH was quickly added dropwise to the mixture₄Aqueous 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 solid product obtained by drying was then dispersed in water (2mg/mL, 5mL) and mixed with L-serine and stirred overnight to give Rh-Ser. Free L-serine was removed by ultrafiltration centrifugation and the purified solution was freeze-dried ready for use. Threonine-modified rhodium nanoenzyme substitutes threonine for serine, and the obtained sample is called Rh-Thr.

FIG. 1 is a scheme showing the synthesis of Rh-Ser in which RhCl is present₃·3H₂O represents rhodium chloride trihydrate and L-Ser represents serine. The Rh-Ser has some ability to target damaged renal tubules.

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

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

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

As shown in FIG. 5, Rh-Ser was able to efficiently scavenge hydroxyl radicals and had a concentration-dependent behavior.

The efficiency of scavenging superoxide anions by varying concentrations of Rh-Ser (0-5. mu.g/mL) was determined by the superoxide dismutase (SOD) detection kit (Sigma-Aldrich, USA). The tests were performed according to the protocol provided by the manufacturer.

As shown in FIG. 6, Rh-Ser was able to scavenge superoxide anions efficiently and had a concentration-dependent profile.

Test for eliminating free radical of 2, 2' -biazobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) by Rh-Ser

The radical scavenging ability of Rh-Ser was determined by ABTS radical cation decolorization. ABTS (7mM) is dissolved in water, and 2.45mM potassium persulfate is added to react for half an hour to generate ABTS free radical cation (ABTS)⁺). Then pure ABTS was determined at 734nm⁺Solution (AB) and different concentrations (0-10. mu.g/mL) of Rh-Ser and ABTS⁺Absorbance value of the mixed solution. The ABTS clearance efficiency is calculated by the formula [ (AB-AP)/AB]*100. All measurements were done in triplicate.

As shown in FIG. 7, Rh-Ser was able to efficiently scavenge ABTS free radicals and had a concentration-dependent behavior.

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

DPPH was used to evaluate the activity of Rh-Ser on scavenging active nitrogen. After mixing Rh-Ser (0-100. mu.g/mL) with different concentrations and 100. mu.M DPPH for 2h, the absorbance (A) of the mixture at 550nm was measured_{Sample (A)}) And absorbance of DPPH at 550nm (A)_{To pair}). The formula for the DPPH removal efficiency is [ (A)_{To pair}-A_{Sample (II)})/A_{To pair}]*100. All measurements were done in triplicate.

As shown in fig. 8, Rh-Ser can effectively scavenge active nitrogen DPPH and has a concentration-dependent characteristic.

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

293T cells or HK-2 cells at 1X 10 per well⁴Density inoculated into 96-well plates and placed at 37 ℃ in 5% CO₂Incubate for 12h under conditions. Next, the old medium in the 96-well plate was aspirated, and medium solutions containing Rh-Ser at different concentrations were added, respectively. After culturing for 20 or 44 hours, the old medium was aspirated from the 96-well plate, and 100. mu.L of MTT medium solution (0.8mg/mL) was added to each well 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: 570nm) of each well was measured on a Synergy H1-type microplate reader using the following formulaAnd calculating the cell survival rate. Cell viability (%) (OD of sample)₅₇₀Value/blank OD₅₇₀Value) × 100%.

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

As shown in FIG. 10, 293T cells were treated with Rh-Ser (0-200. mu.g/mL) 4 hours earlier, cultured for 20 hours with a medium containing 0.5mM hydrogen peroxide, and then the cell viability was determined by the above-described MTT method. Cell viability increased with increasing Rh-Ser concentration, indicating Rh-Ser vs H₂O₂Human tubular epithelial cells (HK-2) under stimulation have a concentration-dependent protective effect.

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

The kidneys of the mice are taken out at different time points respectively, and the kidneys of the mice are imaged by using a photoacoustic imager. As shown in fig. 11, the renal photoacoustic signals of the mice injected with Rh-Ser were significantly enhanced, reaching a peak at 4 hours, which is much higher than those of the other groups, indicating that Rh-Ser has excellent damaged renal targeting ability.

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

All experimental procedures were in accordance with the animal use and health care protocol passed by the animal care and use committee of the clinical centre. Female athymic mice (six weeks, 20-25g) were treated with 8mL/kg of 50% glycerol solution intramuscularly in the hind leg of mice to establish a model of acute renal failure in mice. After 2 hours, small molecular drugs of acetylcysteine or Rh-Ser are injected into tail vein.

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

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

Creatinine and blood urea nitrogen content were measured as shown in FIGS. 13-14. The creatinine and blood urea nitrogen content of Rh-Ser-injected healthy mice did not change significantly. And the creatinine and blood urea nitrogen content of the mice with the injected Rh-Ser acute renal failure is obviously lower than that of the mice with only the injected phosphate buffer, and the levels are close to those of healthy mice. The acetylcysteine with the same dosage can not effectively reduce the two indexes. This shows that Rh-Ser can effectively relieve and treat acute renal failure and has better treatment effect than the small molecule drug acetylcysteine used clinically.

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

In conclusion, the Rh-Ser disclosed by the invention can be used for preparing a large amount of ultra-small nanoparticles through a simple synthesis method, can effectively eliminate various active oxygen/active nitrogen species, and has broad-spectrum active oxygen/active nitrogen elimination capability. The toxic 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 percent after the cells are co-cultured for 24 hours; at the same time they can protect cells from hydrogen peroxide stimulation by scavenging excess reactive oxygen/nitrogen within the cells. Due to the modification of serine, Rh-Ser has the capability of targeting the kidney, and the excellent kidney targeting capability of Rh-Ser is proved by means of a photoacoustic imager. In addition, Rh-Ser shows good treatment effect and excellent anti-inflammatory capability in mice with acute renal failure induced by glycerol. More importantly, Rh-Ser has good biocompatibility and biological safety.

It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.

Claims

1. A nanoenzyme comprising a rhodium nanoparticle and serine bound to the surface of the rhodium nanoparticle.

2. The nanoenzyme of claim 1, wherein the mass ratio of the rhodium nanoparticle to the serine is 1: 1-4.

3. The nanoenzyme of claim 1, wherein the nanoenzyme is a spherical particle having a diameter of less than 5.5 nm.

4. A method for preparing nanoenzyme as claimed in any one of claims 1 to 3, comprising the steps of:

mixing rhodium chloride trihydrate and methoxypolyethylene glycol mercapto in water, adding sodium borohydride after uniform ultrasonic dispersion, and reacting to obtain a rhodium nanoparticle solution;

and (3) carrying out ultrafiltration washing on the rhodium nanoparticle solution, then adding serine, and stirring to combine the serine on the surface of the rhodium nanoparticle, thus obtaining the nano enzyme.

5. The method for preparing nanoenzyme according to claim 4, wherein the mass ratio of the rhodium chloride trihydrate to the methoxypolyethylene glycol mercapto group is 3: 1-10.

6. The method for preparing nanoenzyme according to claim 4, wherein the reaction time is 5-30min in the step of preparing rhodium nanoparticle solution.

7. Use of a nanoenzyme according to any of claims 1 to 3 for the preparation of a medicament for the targeted treatment of acute kidney injury.

8. The use of the nanoenzyme of claim 7, wherein the medicament is in the form of a capsule, tablet, oral preparation, injection, suppository, spray or ointment.