Hybrid nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to the field of biomedicine, particularly provides hybrid nanoparticles as well as a preparation method and application thereof, and aims to solve the adverse effect of excessive lactic acid in circulating blood on a septicemia treatment process. In order to achieve the purpose, the preparation method of the hybrid nano-particles comprises the following steps: preparing hollow mesoporous manganese dioxide modified by polyallylamine hydrochloride; synthesizing the column [5] aromatic hydrocarbon functionalized hyaluronic acid; loading lactate oxidase on the poly (allylamine hydrochloride) modified hollow mesoporous manganese dioxide to obtain an intermediate; and coating the column [5] arene functionalized hyaluronic acid on the surface of the intermediate through electrostatic interaction to obtain the hybrid nanoparticles. The hybrid nanoparticles provided by the invention are simple and convenient to prepare and low in cost, can be widely applied to the fields of materials science, biology, medicine and the like, and have good biocompatibility; the lactic acid concentration of circulating blood can be effectively reduced in a double-pipe lactic acid removal mode, and the LPS-induced septicemia process is expected to be effectively controlled in an in-vivo experiment.

Description

Technical Field

[0001] The present invention relates to the biomedical field, and specifically provides a hybrid nanoparticle and a preparation method and use thereof. Background Art

[0002] Sepsis is a fatal multi-organ failure caused by the host's dysregulated response to infection. It is a life-threatening disease, with approximately 20 million new sepsis patients each year, of which 5.3 million die from sepsis. In the face of the complex physiological and pathological state of sepsis, the current clinical treatment methods are still mainly symptomatic and supportive, including the use of antibiotics, surgery or intervention to eliminate the source of infection; the use of vasoactive drugs, fluid and metabolic resuscitation to stabilize hemodynamics; hemodialysis and immunomodulation to eliminate inflammatory mediators and regulate immune status; mechanical ventilation and extracorporeal oxygenation to obtain organ support and improve organ damage. Although anti-infection treatment and supportive treatment technology have made certain progress in recent years, the in-hospital mortality rate of sepsis is still as high as 25% to 30%, which is one of the main causes of death for patients in intensive care units. In the face of such a life-threatening disease, advanced sepsis treatment strategies are urgently needed to reduce the mortality rate.

[0003] Abnormal lactate accumulation in patients with sepsis has been attributed to increased production by lipopolysaccharide (LPS)-activated neutrophils and systemic tissue hypoxia, as well as reduced metabolic clearance caused by liver and kidney damage. LPS is an important component of the outer membrane of Gram-negative bacteria and a major pathogen-associated molecular pattern, which plays a crucial role in the development and progression of sepsis. After infection, LPS is recognized by the host immune system by binding to Toll-like receptor 4 (TLR4) on the surface of monocytes, macrophages, and neutrophils. Activated neutrophils undergo metabolic reprogramming and enter the anaerobic glycolysis pathway to maintain their function, accompanied by high lactate secretion. Therefore, a dual-pathway approach to clear lactate from circulating blood, from reducing its production to increasing its consumption, may be an effective strategy for treating sepsis. Summary of the invention

[0004] In view of the important role of lactate in the development of sepsis, this patent proposes a lactate regulation strategy to control sepsis. The strategy involves a hollow manganese dioxide nanoparticle loaded with lactate oxidase (LOX) functionalized on the surface of column[5]arene, which consumes existing lactate through LOX and captures LPS-mediated reduction of lactate production through the host-guest interaction of column[5]arene, effectively downregulating the lactate content in circulating blood. Hollow manganese dioxide exhibits catalase-like activity, which can convert hydrogen peroxide generated in the previous reaction into oxygen, providing a guarantee for the continuous consumption of lactate. Therefore, the development of a hybrid nanoparticle that can efficiently remove lactate from circulating blood provides an effective strategy for the treatment of sepsis.

[0005] In order to solve the adverse effects of excessive lactic acid in circulating blood on the treatment process of sepsis, the present invention provides a method for preparing hybrid nanoparticles, comprising the following steps:

[0006] Preparation of hollow mesoporous manganese dioxide modified with polyacrylamine hydrochloride;

[0007] Synthetic pillars [5] Aromatic functionalized hyaluronic acid;

[0008] loading lactate oxidase on the hollow mesoporous manganese dioxide modified by polyacrylamine hydrochloride to obtain an intermediate;

[0009] The column [5] aromatic hydrocarbon functionalized hyaluronic acid is coated on the surface of the intermediate through electrostatic interaction to obtain hybrid nanoparticles.

[0010] In the preparation method of the above technical solution, the preparation of the hollow mesoporous manganese dioxide modified with polyacrylamine hydrochloride specifically comprises the following steps:

[0011] S11: Preparation of silica nanospheres by hydrolysis of tetraethyl orthosilicate;

[0012] S12: Synthesis of hollow mesoporous manganese dioxide by ultrasonic fragmentation;

[0013] S13: using polyacrylamine hydrochloride to perform surface modification on the hollow mesoporous manganese dioxide to obtain the hollow mesoporous manganese dioxide modified with polyacrylamine hydrochloride.

[0014] In the preparation method of the above technical solution, step S11 specifically includes: using a mixture of anhydrous ethanol, ultrapure water and ammonia water as a reaction system, adding tetraethyl orthosilicate at 40° C. and reacting for 12 hours to obtain silica nanospheres;

[0015] And / or, step S12 specifically comprises: using the silicon dioxide nanospheres as a template, adding a potassium permanganate solution dropwise in an ultrasonic environment, continuing the ultrasonication for 6 hours, then centrifuging and washing with water to obtain a transition intermediate, then dispersing the transition intermediate in a sodium carbonate solution for etching reaction, reacting for 12 hours, centrifuging and washing with water to obtain the intermediate;

[0016] And / or, step S13 specifically includes: adding polyacrylamine hydrochloride dropwise into the hollow mesoporous manganese dioxide solution, stirring ultrasonically for 2 hours, centrifuging and washing with water to obtain the hollow mesoporous manganese dioxide modified with polyacrylamine hydrochloride, wherein the solvent in the hollow mesoporous manganese dioxide solution is ethanol.

[0017] In the preparation method of the above technical solution, in the reaction system in step S11, the volume ratio of the anhydrous ethanol, the ultrapure water and the ammonia water is anhydrous ethanol: ultrapure water: ammonia water = 25:0.5:1.8;

[0018] And / or, in step S12, the concentration of the potassium permanganate solution is 3 mg / mL;

[0019] And / or, in step S13, the concentration of the hollow mesoporous manganese dioxide solution is 2 mg / mL.

[0020] In the preparation method of the above technical solution, the synthetic column [5] aromatic hydrocarbon functionalized hyaluronic acid specifically comprises:

[0021] S21: adding L-cysteine ​​methyl ester hydrochloride to the first reaction system and reacting for 24 hours, then dialyzing in a dilute hydrochloric acid solution with a pH of 3.5 for 3 days, and freeze-drying to obtain a first reaction product; wherein the first reaction system is a mixed system of hyaluronic acid, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and deionized water;

[0022] S22: adding the DCM solution containing methacryloyl chloride dropwise to the second reaction system, stirring at 0°C for 2 hours, and then heating to 40°C for 6 hours; after cooling to room temperature, adding 500 mL of water, and then extracting with chloroform three times; washing the organic layer with dilute hydrochloric acid, saturated sodium bicarbonate and water in sequence; drying the organic layer with anhydrous magnesium sulfate and evaporating to dryness to obtain a reaction product 2; wherein the second reaction system is a mixed system of hydroxyethyl-functionalized P[5]A, triethylamine and dichloromethane;

[0023] S23: The reaction product 1 obtained in step S21 is completely dissolved in dimethyl sulfoxide, and dithiothreitol is added and stirred for 1 hour; after the stirring is completed, the reaction product 2 obtained in step S22 and azobisisobutyronitrile are added and reacted for 12 hours, and a nitrogen atmosphere is maintained during the reaction; after the reaction is completed, the obtained solution is thoroughly dialyzed for 3 days and freeze-dried to obtain the column [5] aromatic functionalized hyaluronic acid.

[0024] In the preparation method of the above technical solution, the lactate oxidase is loaded on the hollow mesoporous manganese dioxide modified with polyacrylamine hydrochloride to obtain an intermediate, which specifically includes:

[0025] The polyacrylamine hydrochloride-modified hollow mesoporous manganese dioxide was placed in a clean reaction bottle, lactate oxidase was added, and the mixture was stirred at 4° C. for 30 minutes to obtain the intermediate.

[0026] In the preparation method of the above technical solution, the column [5] aromatic functionalized hyaluronic acid is coated on the surface of the intermediate through electrostatic interaction to obtain hybrid nanoparticles, which specifically includes:

[0027] The column [5] aromatic functionalized hyaluronic acid is added to the intermediate, stirred at 4° C. for 15 minutes, and then centrifuged and washed to collect nanoparticles, which are then washed with ultrapure water to obtain the hybrid nanoparticles.

[0028] The present invention also provides a hybrid nanoparticle, which is prepared by the method for preparing the hybrid nanoparticle described in any one of the technical solutions above.

[0029] The present invention also provides a use of the hybrid nanoparticles as described above for capturing LPS and clearing lactic acid in treating sepsis.

[0030] The beneficial effects of the present invention are as follows:

[0031] In the case of adopting the above technical scheme, the present invention can capture the synergistic effect of LPS-mediated lactic acid production reduction through the host-guest interaction of LOX consuming the produced lactic acid and column [5] aromatics, and effectively reduce the lactic acid content in the blood. The hollow manganese dioxide exhibits catalase-like activity, converting the hydrogen peroxide generated in the previous reaction into oxygen, providing a guarantee for continuous lactic acid consumption.

[0032] The hybrid nanoparticles provided by the present invention are easy to prepare and low in cost, can be widely used in the fields of materials science, biology, medicine, etc., have good biocompatibility, and can effectively reduce the circulating blood lactate concentration in a two-pronged lactate clearance manner, thereby inhibiting the induced high vascular permeability and alleviating macrophage-related systemic inflammatory response, and are expected to effectively control the process of LPS-induced sepsis in in vivo experiments.

[0033] The hybrid nanoparticles are used to capture LPS and remove lactic acid in the treatment of diseases, and can efficiently remove lactic acid in circulating blood, providing an effective strategy for the treatment of sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings, in which:

[0035] Figure 1 is the H NMR spectrum of HA-SH in Example 2;

[0036] Figure 2 is the H NMR spectrum of MMAP[5]A in Example 2;

[0037] Figure 3 is the H NMR spectrum of HA-P[5]A in Example 2;

[0038] Figure 4 is a transmission electron microscope image of LOX@HMnO2-P[5]A in Example 4;

[0039] Figure 5 is a quantitative data graph of hydrogen peroxide consumption in Example 5;

[0040] Figure 6 is a quantitative data graph of oxygen generation in Example 5;

[0041] Figure 7 is a quantitative data graph of lactic acid produced by neutrophils in Example 5;

[0042] Figure 8 is a quantitative data diagram of inflammatory factors secreted by macrophages in Example 5;

[0043] Fig. 9 is a graph of the lactic acid content in circulating blood in Example 6;

[0044] Fig.10 is a graph of LPS content in circulating blood in Example 6;

[0045] Fig.11 The figure is a flow chart of a method for preparing hybrid nanoparticles according to an embodiment. DETAILED DESCRIPTION

[0046] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the protection scope of the present invention.

[0047] The present invention provides a method for preparing hybrid nanoparticles. Fig.11 , Fig.11 The figure is a flow chart of a method for preparing hybrid nanoparticles according to an embodiment.

[0048] The method comprises the following steps:

[0049] Step S1, preparing hollow mesoporous manganese dioxide modified with polyacrylamine hydrochloride;

[0050] Step S2, synthesizing column [5] aromatic functionalized hyaluronic acid;

[0051] Step S3, loading lactate oxidase on the hollow mesoporous manganese dioxide modified with polyacrylamine hydrochloride to obtain an intermediate;

[0052] Step S4, coating the column [5] aromatic functionalized hyaluronic acid on the surface of the intermediate through electrostatic interaction to obtain hybrid nanoparticles.

[0053] In Example 1, the preparation of the hollow mesoporous manganese dioxide modified with polyacrylamine hydrochloride specifically includes the following steps S11 to S13:

[0054] This embodiment takes the preparation of a modified hollow mesoporous manganese dioxide (PAH--HMnO2) as an example, and includes the following steps:

[0055] Step S11: preparing silica nanospheres through the hydrolysis reaction of tetraethyl orthosilicate.

[0056] Specifically, the method comprises: using a mixture of anhydrous ethanol, ultrapure water and ammonia water as a reaction system, adding tetraethyl orthosilicate at 40° C. and reacting for 12 hours to obtain silicon dioxide nanospheres.

[0057] First, using the hydrolysis reaction of tetraethyl orthosilicate, 25 mL of anhydrous ethanol, 0.5 mL of ultrapure water, and 1.8 mL of ammonia water were added to the reaction bottle, mixed and stirred evenly, and placed in a 40°C constant temperature oil bath as the reaction environment liquid for the hydrolysis reaction of tetraethyl orthosilicate. The temperature was kept at 40°C for 10 minutes, and 0.75 mL of tetraethyl orthosilicate was quickly added dropwise to the reaction liquid. After 12 hours, silica nanospheres were obtained.

[0058] In the reaction system in step S11, the volume ratio of the anhydrous ethanol, the ultrapure water and the ammonia water is anhydrous ethanol: ultrapure water: ammonia water = 25:0.5:1.8.

[0059] Step S12: Synthesize hollow mesoporous manganese dioxide (HMnO2) by ultrasonic crushing.

[0060] Specifically, the process includes: using the silicon dioxide nanospheres as a template, dropping a potassium permanganate solution in an ultrasonic environment, continuing the ultrasonication for 6 hours, then centrifuging and washing with water to obtain a transition intermediate, then dispersing the transition intermediate in a sodium carbonate solution for an etching reaction, and after reacting for 12 hours, centrifuging and washing with water to obtain the intermediate.

[0061] Wherein, the concentration of the potassium permanganate solution is 3 mg / mL.

[0062] Specifically, hollow mesoporous manganese dioxide was synthesized by ultrasonic crushing, and the silica nanospheres were used as templates. 50 mL (3 mg / mL) of potassium permanganate solution was added dropwise in an ultrasonic environment, and the ultrasound was continued for 6 hours. After the reaction was completed, SiO2@HMnO2 was obtained by centrifugation (11000 rpm, 15 minutes) and washing with water 3 times. Finally, SiO2@HMnO2 was dispersed in 20 mL (2 mol / L) of sodium carbonate solution to react for 12 hours to etch away the silica nanospheres. After the reaction was completed, the silica nanospheres were centrifuged (11000 rpm, 15 minutes) and washed with water 3 times to obtain hollow mesoporous manganese dioxide.

[0063] Step S13: using polyacrylamine hydrochloride (PAH) to perform surface modification on the hollow mesoporous manganese dioxide to obtain the hollow mesoporous manganese dioxide modified with polyacrylamine hydrochloride.

[0064] Specifically, it comprises: adding polyacrylamine hydrochloride dropwise into the hollow mesoporous manganese dioxide solution, stirring ultrasonically for 2 hours, centrifuging and washing with water to obtain the hollow mesoporous manganese dioxide modified with polyacrylamine hydrochloride, wherein the solvent in the hollow mesoporous manganese dioxide solution is ethanol.

[0065] Wherein, the concentration of the hollow mesoporous manganese dioxide solution is 2 mg / mL.

[0066] In order to improve the stability of the HMnO2, PAH was introduced to modify its surface. The cationic PAH was coated on the negatively charged surface of HMnO2 by electrostatic interaction, and 10 mL (5 mg / mL) of PAH was added dropwise into 5 mL (2 mg / mL) of hollow mesoporous manganese dioxide solution, and ultrasonic stirring was performed for 2 hours. After the reaction, the solution was centrifuged and washed with water for 3 times to obtain PAH-HMnO2.

[0067] Embodiment 2:

[0068] This example takes the synthesis of a pillar[5]arene functionalized hyaluronic acid (HA-P[5]A) as an example, and includes the following steps:

[0069] S21: L-cysteine ​​methyl ester hydrochloride is added to the first reaction system to react for 24 hours, then thoroughly dialyzed in a dilute hydrochloric acid solution with a pH of 3.5 for 3 days, and freeze-dried to obtain a first reaction product (HA-SH); wherein the first reaction system is a mixed system of hyaluronic acid, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and deionized water.

[0070] Specifically, hyaluronic acid (HA) (1g, Mw 30-40kDa) and N-hydroxysuccinimide (1.15g, 10mmol) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (1.916g, 10mmol) are dissolved in 100mL deionized water and placed in a 250mL reaction bottle. After stirring at room temperature for 1 hour, the carboxyl group of hyaluronic acid is fully activated, and then L-cysteine ​​methyl ester hydrochloride (1.710g, 10mmol) is added to the above-mentioned reaction solution, and the reaction is continued for 24 hours under dark conditions. Throughout the process, the pH value of the solution is 4.8 by adding 1M hydrochloric acid. After the reaction is completed, it is thoroughly dialyzed (MWCO, 3500Da) for 3 days in a dilute hydrochloric acid solution with a pH of 3.5, and freeze-dried to obtain HA-SH.

[0071] like Figure 1As shown, the H-NMR spectrum data of HA and HA-SH: the characteristic peak of HA-SH appears at about 2.2-2.8 ppm.

[0072] S22: Add the DCM solution containing methacryloyl chloride dropwise to the second reaction system, stir at 0°C for 2 hours, and then heat to 40°C for reaction for 6 hours; after cooling to room temperature, add 500 mL of water, and then extract with chloroform three times; wash the organic layer with dilute hydrochloric acid, saturated sodium bicarbonate and water in turn; dry the organic layer with anhydrous magnesium sulfate and evaporate to dryness to obtain reaction product II; wherein the second reaction system is a mixed system of hydroxyethyl-functionalized P[5]A, triethylamine and dichloromethane.

[0073] Specifically, MC2HP[5]A (hydroxyethyl functionalized P[5]A) (850 mg, 1.11 mM), triethylamine (TEA, 202.4 mg, 2 mM) and dichloromethane (DCM, 20 mL) were added to a two-necked flask in an ice-water bath, and the mixture was stirred for 30 minutes. A DCM (20 mL) solution containing methacryloyl chloride (139.4 mg, 1.33 mM) was added dropwise to the above solution within 2 hours. Stirring was continued at 0°C for 2 hours, and then heated to 40°C for 6 hours. After cooling to room temperature, 500 mL of water was added, and then extracted with chloroform three times. The organic layer was washed with dilute hydrochloric acid, saturated sodium bicarbonate and water in sequence. The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness to obtain the pure product of MMAP[5]A as a light orange powder.

[0074] like Figure 2 As shown, H NMR data indicate the successful synthesis of MMAP[5]A.

[0075] S23: The reaction product 1 obtained in step S21 is completely dissolved in dimethyl sulfoxide, and dithiothreitol is added and stirred for 1 hour; after the stirring is completed, the reaction product 2 obtained in step S22 and azobisisobutyronitrile are added and reacted for 12 hours, and a nitrogen atmosphere is maintained during the reaction; after the reaction is completed, the obtained solution is thoroughly dialyzed for 3 days and freeze-dried to obtain the column [5] aromatic functionalized hyaluronic acid.

[0076] Specifically, the HA-SH (1 g, Mw 30-40 kDa) was completely dissolved in dimethyl sulfoxide (DMSO), and the reducing agent dithiothreitol (DTT) was added and stirred for 1 hour to prevent thiol cross-linking. After the stirring was completed, the MMAP[5]A described in step S22 and the initiator azobisisobutyronitrile (AIBN) were added to react for 12 hours, and a nitrogen atmosphere was maintained during the reaction. After the reaction was completed, the resulting solution was thoroughly dialyzed (MWCO, 3500 Da) for 3 days and freeze-dried to obtain the target product HA-P[5]A.

[0077] like Figure 3As shown, H NMR data indicated the successful synthesis of HA-P[5]A.

[0078] Embodiment 3:

[0079] This embodiment takes the loading of lactate oxidase (LOX) on the PAH-HMnO2 to obtain LOX@HMnO2 as an example, and includes the following steps:

[0080] PAH-HMnO2 solution (1.8 mL, 1 mg / mL) was placed in a clean reaction bottle, LOX (200 μL, 1 mg / mL) was added, and stirred at 4°C for 30 minutes to obtain LOX@HMnO2.

[0081] Example 4

[0082] This embodiment takes the electrostatic interaction coating of HA-P[5]A on LOX@HMnO2 as an example, and includes the following steps:

[0083] HA-P[5]A (1:4) was added to LOX@HMnO2 and stirred at 4°C for 15 minutes. After the reaction was completed, the nanoparticles were collected by centrifugal washing (10000 rpm, 10 minutes), and then washed with ultrapure water for 3 times to obtain hybrid nanoparticles LOX@HMnO2-P[5]A that were stably stored in water.

[0084] Comparative Example 1

[0085] HA-P[5]A (1:2) was added to LOX@HMnO2 and stirred at 4°C for 15 minutes. After the reaction was completed, the nanoparticles were collected by centrifugal washing (10000 rpm, 10 minutes), and then washed with ultrapure water for 3 times to obtain hybrid nanoparticles LOX@HMnO2-P[5]A that were stably stored in water.

[0086] Example 5

[0087] Various performance tests were performed on the above LOX@HMnO2-P[5]A. The specific experiments are as follows:

[0088] 1. Consume hydrogen peroxide and generate oxygen.

[0089] In order to verify that the hydrogen peroxide produced by LOX consuming oxygen can be consumed to generate oxygen to further oxidize lactic acid, LOX@HMnO2-P[5]A was incubated with sodium lactate, and the concentration of hydrogen peroxide at different times was detected using a hydrogen peroxide detection kit, and the generation of oxygen was measured using a dissolved oxygen meter.

[0090] Comparative Example 2

[0091] In order to verify that the hydrogen peroxide produced by LOX consuming oxygen can be consumed to generate oxygen to further oxidize lactic acid, LOX was incubated with sodium lactate, and the concentration of hydrogen peroxide at different times was detected using a hydrogen peroxide detection kit, and the generation of oxygen was measured using a dissolved oxygen meter.

[0092] like Figure 5 As shown, as the reaction proceeds, the concentration of hydrogen peroxide gradually decreases. This is because the hydrogen peroxide produced by the oxidation of lactic acid is catalyzed by HMnO2 to produce oxygen to further oxidize lactic acid.

[0093] like Figure 6 As shown, LOX@HMnO2-P[5]A significantly reduced the oxygen consumption.

[0094] 3. Capturing LPS inhibits neutrophil lactate production

[0095] Neutrophils (1×10 6 ) were seeded on 24-well plates in serum-free 1640 medium and then stimulated in groups: (1) PBS (control), (2) LPS (untreated), (3) LPS+LOX, (4) LPS+LOX@HA-HMnO2, (5) LPS+HMnO2-P[5]A, (6) LPS+LOX@HMnO2-P[5]A for 6 hours. Cell-free supernatants from non-stimulated or LPS-stimulated cultured neutrophils were harvested at 5 hours and analyzed for lactate content using a lactate detection kit.

[0096] like Figure 7 As shown, an increase in lactate production by LPS-stimulated BM neutrophils was observed. In contrast, treatment with LOX or LOX@HA-HMnO2 significantly reduced lactate production by LPS-stimulated BM neutrophils, indicating that LOX plays an important role in reducing lactate.

[0097] 4. Blocking LPS-induced macrophage inflammatory response

[0098] RAW 264.7 cells were seeded in 6-well plates at 50,000 cells / well and cultured for 24 hours at 37°C and 5% CO2. The cells were then cultured with LPS and hybrid nanoparticles for 24 hours. The inflammatory factor tumor necrosis factor-α (TNF-α) detection kit was used to analyze the inflammatory factor content.

[0099] like Figure 8 As shown, LPS also significantly induced the expression of tumor necrosis factor-α (TNF-α), and after treatment with LOX@HMnO2-P[5]A, TNF-α expression was significantly inhibited.

[0100] In another aspect, the present invention further provides a hybrid nanoparticle, wherein the hybrid nanoparticle is prepared by the method for preparing the hybrid nanoparticle described in any one of the technical solutions above.

[0101] like Figure 1 As shown in Figures 2 and 3, the H NMR spectra characterized the successful synthesis of HA-SH, MMAP[5]A, and HA-P[5]A.

[0102] like Figure 4 As shown, the transmission electron microscopy image characterizes the morphology of LOX@HMnO2-P[5]A.

[0103] Furthermore, the present invention also provides a use of the hybrid nanoparticles as described above for capturing LPS and clearing lactic acid in treating diseases.

[0104] Example 6

[0105] The LOX@HMnO2-P[5]A was subjected to a circulating blood lactate and LPS clearance experiment. The specific experiment is as follows:

[0106] After the LPS-induced sepsis mouse model was successfully established, the mice were randomly divided into 6 groups (n=9): (1) PBS, (2) LPS+PBS, (3) LPS+LOX, (4) LPS+LOX@HA-HMnO2, (5) LPS+HMnO2-P[5]A, (6) LPS+LOX@HMnO2-P[5]A. Mice were treated with intravenous injection of groups (1)-(6) 1 hour after LPS injection, and the drug was administered three times within 48 hours. After treatment, orbital blood samples were collected to collect serum, and the concentrations of lactate and LPS were quantified.

[0107] like Fig. 9 As shown, all treatments resulted in a significant decrease in blood lactate levels, and LOX@HMnO2-P[5]A reduced circulating blood lactate levels more significantly.

[0108] like Fig.10 As shown, the LOX@HMnO2-P[5]A-treated group significantly reduced the LPS content in circulating blood, indicating that LOX@HMnO2-P[5]A has excellent LPS scavenging ability.

[0109] Another aspect of the present invention further provides a hybrid nanoparticle, wherein the hybrid nanoparticle is prepared by the preparation method of the hybrid nanoparticle described in any one of the technical solutions above.

[0110] The hybrid nanoparticles provided by the present invention are easy to prepare and low in cost, can be widely used in the fields of materials science, biology, medicine, etc., have good biocompatibility, and can effectively reduce the circulating blood lactate concentration in a two-pronged lactate clearance manner, thereby inhibiting the induced high vascular permeability and alleviating macrophage-related systemic inflammatory response, and are expected to effectively control the process of LPS-induced sepsis in in vivo experiments.

[0111] Furthermore, the present invention also provides a use of the hybrid nanoparticles as described above for capturing LPS and clearing lactic acid in the treatment of sepsis.

[0112] In the case of adopting the above technical scheme, the present invention can capture the synergistic effect of LPS-mediated lactic acid production reduction through the host-guest interaction of LOX consuming the produced lactic acid and column [5] aromatics, and effectively reduce the lactic acid content in the blood. The hollow manganese dioxide exhibits catalase-like activity, converting the hydrogen peroxide generated in the previous reaction into oxygen, providing a guarantee for continuous lactic acid consumption.

[0113] The hybrid nanoparticles are used to capture LPS and remove lactic acid in the treatment of diseases, and can efficiently remove lactic acid in circulating blood, providing an effective strategy for the treatment of sepsis.

Claims

1. A method for preparing hybrid nanoparticles, characterized in that: The following steps are involved: Preparation of hollow mesoporous manganese dioxide modified with polyacrylamine hydrochloride; Synthetic pillars [5] Aromatic functionalized hyaluronic acid; loading lactate oxidase on the hollow mesoporous manganese dioxide modified by polyacrylamine hydrochloride to obtain an intermediate; The column [5] aromatic hydrocarbon functionalized hyaluronic acid is coated on the surface of the intermediate through electrostatic interaction to obtain hybrid nanoparticles.

2. The method for preparing hybrid nanoparticles according to claim 1, characterized in that: The preparation of the hollow mesoporous manganese dioxide modified with polyacrylamine hydrochloride specifically comprises the following steps: S11: Preparation of silica nanospheres by hydrolysis of tetraethyl orthosilicate; S12: Synthesis of hollow mesoporous manganese dioxide by ultrasonic fragmentation; S13: using polyacrylamine hydrochloride to perform surface modification on the hollow mesoporous manganese dioxide to obtain the hollow mesoporous manganese dioxide modified with polyacrylamine hydrochloride.

3. The method for preparing hybrid nanoparticles according to claim 2, characterized in that: Step S11 specifically includes: using a mixture of anhydrous ethanol, ultrapure water and ammonia water as a reaction system, adding tetraethyl orthosilicate to react for 12 hours at 40° C. to obtain silica nanospheres; And / or, step S12 specifically includes: using the silicon dioxide nanospheres as a template, adding a potassium permanganate solution dropwise in an ultrasonic environment, continuing the ultrasonication for 6 hours, then centrifuging and washing with water to obtain a transition intermediate, then dispersing the transition intermediate in a sodium carbonate solution for etching reaction, reacting for 12 hours, then centrifuging and washing with water to obtain hollow mesoporous manganese dioxide; And / or, step S13 specifically includes: adding polyacrylamine hydrochloride dropwise into the hollow mesoporous manganese dioxide solution, stirring ultrasonically for 2 hours, centrifuging and washing with water to obtain the hollow mesoporous manganese dioxide modified with polyacrylamine hydrochloride, wherein the solvent in the hollow mesoporous manganese dioxide solution is ethanol.

4. The method for preparing hybrid nanoparticles according to claim 3, characterized in that: In the reaction system in step S11, the volume ratio of the anhydrous ethanol, the ultrapure water and the ammonia water is anhydrous ethanol: ultrapure water: ammonia water = 25:0.5:1.8; And / or, in step S12, the concentration of the potassium permanganate solution is 3 mg / mL; And / or, in step S13, the concentration of the hollow mesoporous manganese dioxide solution is 2 mg / mL.

5. The method for preparing hybrid nanoparticles according to claim 1, characterized in that: The synthetic column [5] aromatic hydrocarbon functionalized hyaluronic acid specifically comprises: S21: adding L-cysteine ​​methyl ester hydrochloride to the first reaction system and reacting for 24 hours, then dialyzing in a dilute hydrochloric acid solution with a pH of 3.5 for 3 days, and freeze-drying to obtain a first reaction product; wherein the first reaction system is a mixed system of hyaluronic acid, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and deionized water; S22: adding the DCM solution containing methacryloyl chloride dropwise to the second reaction system, stirring at 0°C for 2 hours, and then heating to 40°C for 6 hours; after cooling to room temperature, adding 500 mL of water, and then extracting with chloroform three times; washing the organic layer with dilute hydrochloric acid, saturated sodium bicarbonate and water in sequence; drying the organic layer with anhydrous magnesium sulfate and evaporating to dryness to obtain a reaction product 2; wherein the second reaction system is a mixed system of hydroxyethyl-functionalized P[5]A, triethylamine and dichloromethane; S23: The reaction product 1 obtained in step S21 is completely dissolved in dimethyl sulfoxide, and dithiothreitol is added and stirred for 1 hour; after the stirring is completed, the reaction product 2 obtained in step S22 and azobisisobutyronitrile are added and reacted for 12 hours, and a nitrogen atmosphere is maintained during the reaction; after the reaction is completed, the obtained solution is thoroughly dialyzed for 3 days and freeze-dried to obtain the column [5] aromatic functionalized hyaluronic acid.

6. The method for preparing hybrid nanoparticles according to claim 1, characterized in that: The method of loading lactate oxidase on the hollow mesoporous manganese dioxide modified with polyacrylamine hydrochloride to obtain an intermediate specifically comprises: The polyacrylamine hydrochloride-modified hollow mesoporous manganese dioxide was placed in a clean reaction bottle, lactate oxidase was added, and the mixture was stirred at 4° C. for 30 minutes to obtain the intermediate.

7. The method for preparing hybrid nanoparticles according to claim 1, characterized in that: The process of coating the column [5] aromatic functionalized hyaluronic acid on the surface of the intermediate through electrostatic interaction to obtain hybrid nanoparticles specifically comprises: The column [5] aromatic functionalized hyaluronic acid is added to the intermediate, stirred at 4° C. for 15 minutes, and then centrifuged and washed to collect nanoparticles, which are then washed with ultrapure water to obtain the hybrid nanoparticles.

8. A hybrid nanoparticle, characterized in that: The hybrid nanoparticles are prepared by the preparation method described in any one of claims 1 to 7.

9. Use of the hybrid nanoparticles according to claim 8 for capturing LPS and scavenging lactate in the treatment of sepsis.

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