Multifunctional biomimetic nanoparticles with an enzyme as a targeting functional motif, preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a multifunctional biomimetic nanoparticle taking an enzyme as a targeting functional motif, a preparation method and application. The application obtains a cell membrane-coated drug-loaded dopamine nanoparticle through the following steps: synthesizing a PDA nanoparticle solution, drug loading, membrane coating, biotin-modified enzyme and streptavidin reaction, and the cell membrane-coated drug-loaded dopamine nanoparticle has strong compatibility and good stability, is suitable for selective delivery of hydrophobic drugs, can effectively deliver drugs to a lesion site, prolongs the circulation time of the enzyme, protects the activity of the enzyme in the body, relieves oxidative stress, and improves the drug concentration at the lesion site, can effectively regulate ischemic stroke neurovascular units, degrade NETs, relieve oxidative stress, improve neuron vitality, and inhibit glial cell activation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a multifunctional biomimetic nanoparticle with an enzyme as a target functional motif, its preparation method, and its application. Background Technology

[0002] Compared to traditional small-molecule drugs, enzymes possess higher specificity and bioactivity, offering immense potential for disease treatment. The complexity of biomacromolecules endows them with greater specificity and efficacy, but also presents challenges in stability and delivery across biological barriers. Various drug delivery systems have been developed to protect enzyme activity, prolong cycle time, and deliver enzymes to specific regions. On the other hand, enzymes are also used as functional modules in drug delivery systems. Through asymmetric modification of enzymes, nanomotors exhibit chemotactic behavior with self-navigation and self-targeting capabilities across substrate concentration gradients in disease-related regions. In sequential delivery, local release of enzymes has also been used to clear delivery barriers. These explorations expand the application of enzymes in drug delivery systems, but remain limited to their catalytic role. While the driving forces of enzymes have been reported, suitable applications in the disease microenvironment remain elusive.

[0003] Acute ischemic stroke is one of the most common cerebrovascular diseases, with high mortality and severe disability. Although several standard thrombolytic therapies, represented by recombinant tissue plasminogen activator (rt-PA), have been developed, severe secondary ischemia-reperfusion injury leads to poor prognosis in clinical practice. As a key component of the cerebrovascular system, the neurovascular unit (NVU) is affected by the large amounts of reactive oxygen species (ROS) generated during reperfusion, leading to structural and functional damage. NVU homeostasis is disrupted, including neuronal apoptosis, abnormal glial cell activation, endothelial cell damage, loosening of tight junctions, and disruption of the blood-brain barrier (BBB) ​​integrity. Increasing evidence suggests that neutrophils play a crucial role in ischemia-reperfusion injury. Neutrophils can be rapidly recruited to the lesion area and overactivated, forming neutrophil extracellular traps (NETs). Released proteases and chromatin may exacerbate neurotoxicity and inflammatory cascades. Reticulum DNA acts as a microthrombus backbone, trapping other blood cells and forming immune thrombi. Based on the complex structure and functional crosstalk of the cerebral vascular network, multi-target therapeutic strategies that remodel neurovascular units may be a promising treatment for effectively alleviating reperfusion injury. Summary of the Invention

[0004] To address the aforementioned problems, this invention, based on existing technological foundations, provides a multifunctional biomimetic nanoparticle with an enzyme as a targeting functional motif, along with its preparation method and applications. This invention uses deoxyribonuclease I (DNase I) to target nanoparticles to NET-rich ischemia-reperfusion regions; combining the therapeutic and targeting functions of the enzyme simplifies the design and preparation of drug delivery systems, and improves their efficacy and clinical translational value.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing multifunctional biomimetic nanoparticles with an enzyme as a target functional motif, the specific steps of which are as follows: (1) Dopamine hydrochloride was subjected to oxidative polymerization at room temperature to obtain a polydopamine (PDA) nanoparticle solution; (2) Stir the polydopamine nanoparticle solution and the drug obtained in step (1) at room temperature to obtain a drug-loaded nanoparticle solution; (3) After mixing the drug-loaded nanoparticles and the membrane described in step (2), the mixture is extruded or sonicated to obtain a coated nanoparticle solution; (4) The coated nanoparticles described in step (3) are co-incubated with DSPE-PEG-AAPVK-biotin at room temperature to obtain a solution of coated nanoparticles with biotin exposed on the surface by responsive peptides. The DSPE-PEG-AAPVK-biotin is a block copolymer of neutrophil elastase, namely 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-poly(ethylene glycol)-alsnine-alsnine-proline-valine-lysine-biotin (DSPE-PEG-AAPVK-biotin). The nanoparticles are connected to enzymes via neutrophil elastase-responsive sequences, which accumulate in the NETs region and release enzymes in response, thereby increasing the drug concentration at the lesion site.

[0006] (5) The enzyme is reacted with biotin succinimide ester at room temperature to obtain a biotin-modified enzyme, wherein the enzyme is an enzyme with an amino group that does not affect catalytic activity. (6) First, streptavidin is mixed with the biotin-modified enzyme and stirred at room temperature. Then, it is added to the nanoparticle solution obtained in step (4) and left to stand at room temperature to obtain multifunctional biomimetic nanoparticles with enzyme as the target functional motif. Preferably, in step (1), pure water, anhydrous ethanol, and ammonia are mixed at room temperature and stirred for 30 minutes. Then, an aqueous solution of dopamine hydrochloride is added, and stirring is continued for 24 hours with the mixture exposed to the open. Organic solvents and unpolymerized raw materials are removed by ultrafiltration to obtain a polydopamine nanoparticle solution. The volume ratio of pure water, anhydrous ethanol, and ammonia is 1:(0.4~0.6):(0.08~0.1), the mass concentration of ammonia is 25%~28%, the concentration of the aqueous solution of dopamine hydrochloride is 50 mg / mL, and the mass-volume ratio of dopamine hydrochloride to pure water is (4~6):1, mg / mL.

[0007] Preferably, in step (2), the drug is first dissolved in an organic solvent and then added to a polydopamine nanoparticle solution and stirred at room temperature for 12 hours; preferably, the mass ratio of the drug to the polydopamine nanoparticles is 1:(4~10), the drug is a hydrophobic drug, preferably idebenone, curcumin, retinol or rapamycin, and more preferably idebenone, and the organic solvent is dimethyl sulfoxide.

[0008] Preferably, the membrane in step (3) is a cell membrane or a mixed membrane; the mass ratio of the membrane to the drug-loaded nanoparticles is 1:(1~2); the ultrasonic temperature is 4°C and the ultrasonic time is 2 minutes.

[0009] Preferably, in step (4), the coated nanoparticles obtained in step (3) are mixed with DSPE-PEG-AAPVK-biotin and incubated together at room temperature for 1 hour; the DSPE-PEG-AAPVK-biotin was purchased from Jier Biochemical (Shanghai) Co., Ltd.; the mass ratio of the coated nanoparticles obtained in step (3) to DSPE-PEG-AAPVK-biotin is 1: (0.002~0.005).

[0010] Preferably, in step (5), the enzyme is first dissolved in phosphate buffer, biotin succinimide (NHS-biotin) is dissolved in dimethyl sulfoxide, the biotin succinimide solution is added to the enzyme solution, and the reaction is stirred at room temperature for 1 hour; the enzyme is deoxyribonuclease I; the molar ratio of the enzyme to biotin succinimide is 1: (0.8~1.2).

[0011] Preferably, in step (6), the stirring reaction time is 1 hour and the standing time is 1 hour; the molar ratio of streptavidin, biotin coupled with the coated nanoparticles in step (4) and biotin coupled with the enzyme in step (5) is 1:(1~3):(1~3).

[0012] The present invention also includes the application of multifunctional biomimetic nanoparticles with enzymes obtained by the above preparation method as targeting functional motifs in multi-target regulation of ischemia-reperfusion injury and targeted delivery of NETs.

[0013] This invention utilizes DNase I to target nanoparticles to ischemia-reperfusion regions rich in NETs. The nanoparticles are linked to the surface of dopamine nanoparticles coated on the erythrocyte membrane via cleavable peptides, thereby prolonging circulation time. Based on the chemotaxis of DNA due to concentration gradients and interactions with DNA, the nanoparticles accumulate in the lesion region. Simultaneously, cleavage by high concentrations of neutrophil elastase (NE) releases DNase I to degrade pathological NETs and allows the nanoparticles to enter the brain via the damaged BBB for multi-target therapy.

[0014] Compared with the prior art, the present invention has the following advantages: (1) The nanoparticle raw materials of the present invention are simple and readily available, easy to prepare, highly compatible, and have good stability, which is conducive to clinical translation and mass production.

[0015] (2) Compared with the traditional intravenous administration method, the nanoparticles of the present invention have obvious targeting effect on brain lesion areas, strong specificity and high delivery efficiency.

[0016] (3) The nanoparticles of the present invention can achieve multi-target therapy through the co-delivery of enzymes, drugs and functional carriers, comprehensively regulate the internal and external microenvironment of NVU, degrade pathological NETs, ​​protect the blood-brain barrier and neurons, and reduce ischemia-reperfusion injury. Attached Figure Description

[0017] Figure 1 This is a roadmap for nanoparticle preparation. Figure 2 Let be the particle size and electric potential of the nanoparticles, where Figure 2 A is the particle size distribution diagram. Figure 2 B is the particle size distribution chart. Figure 2 C represents the surface potential statistics of nanoparticles; Figure 3 The graph shows the ROS scavenging ability of polydopamine nanoparticles. Figure 3 A is the UV spectrum after co-incubation with DPPH· Figure 3 B is a statistical chart of residual DPPH· after co-incubation with DPPH·; Figure 4 The images show the in vivo and brain distribution of nanoparticles in tMCAO model mice and sham-operated mice after injection. Figure 4 A shows the fluorescence distribution of the major organs. Figure 4 B is a fluorescence distribution map in the brain. Figure 4 C is the TTC staining pattern; Figure 5 To investigate the effectiveness of nanoparticles in removing NETs; Figure 6 To investigate the neuroprotective effect of nanoparticles; Figure 7 The image shows the therapeutic effect of injecting nanoparticles into tMCAO model mice. Figure 7 Image A shows the results of TTC staining. Figure 7 B is a statistical chart of infarct area. Detailed Implementation

[0018] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0019] Example 1 A method for preparing multifunctional biomimetic nanoparticles with an enzyme as a target functional motif, such as... Figure 1 As shown, it includes the following steps: (1) Synthesis of PDA nanoparticle solution: 9 mL of pure water, 4 mL of anhydrous ethanol and 800 μL of ammonia water were added to the reaction flask and stirred at room temperature for 30 minutes; 50 mg of dopamine hydrochloride was weighed, dissolved in 1 mL of pure water, added to the reaction flask, and stirred open at room temperature for 24 hours; after the reaction was completed, the reaction solution was ultrafiltered multiple times and filtered using a 0.22 μm microporous membrane to obtain purified PDA nanoparticle solution.

[0020] (2) Drug loading: Weigh idebenone and dissolve it in dimethyl sulfoxide. Add idebenone and PDA nanoparticles in a mass ratio of 1:4 to the PDA nanoparticle solution obtained in step (1). Stir at room temperature for 12 hours, centrifuge and take the supernatant to obtain drug-loaded PDA nanoparticles PDA / IDB.

[0021] (3) Red blood cell membrane coating: Mouse red blood cell membrane and PDA nanoparticles obtained in step (2) were mixed at a mass ratio of 1:2 and sonicated at 4°C for 2 minutes to obtain red blood cell membrane coated nanoparticles RM@(PDA / IDB).

[0022] (4) Modification of DNase I: The coated nanoparticles were mixed with DSPE-PEG-AAPVK-biotin and allowed to stand at room temperature for 1 hour to obtain a solution of coated nanoparticles with biotin exposed on the surface by responsive peptides; DNase I was dissolved in phosphate buffer, biotin succinimide was dissolved in dimethyl sulfoxide, and the biotin succinimide solution was added to the DNase I solution at a molar ratio of 1:1 and stirred at room temperature for 1 hour; streptavidin was mixed with the biotin-modified enzyme, and the molar ratio of streptavidin to biotin coupled with DNase I was 1:3, and stirred at room temperature for 1 hour to obtain SA-3DNase; SA-3DNase was added to the solution of coated nanoparticles with biotin exposed on the surface by responsive peptides, and the molar ratio of SA-3DNase to biotin coupled with coated nanoparticles was 1:1, and allowed to stand at room temperature for 1 hour to obtain the final nanoparticle DNase-RM@(PDA / IDB).

[0023] The particle size and potential of nanoparticles are measured using a particle size analyzer, such as... Figure 2 As shown: the particle size of each group of nanoparticles is approximately 100 nm. After enzyme modification, the particle size increases and the potential is approximately -25 mV, making it suitable for intravenous administration.

[0024] The ability of PDA nanoparticles to scavenge ROS was investigated using the DPPH method, such as Figure 3 As shown: the fluorescence decreased after co-incubation, indicating that free radicals were cleared; the higher the PDA concentration, the stronger the antioxidant capacity.

[0025] Primary mouse neutrophils were extracted, and neutrophils were induced to form NETs using phorbol 12-tetradecanoate 13-acetate (PMA). After treatment with DNase-RM@ (PDA / IDB), NET-related proteins were immunofluorescently stained as shown below. Figure 5 As shown: After PMA induction, an increase in NETs markers (citrullinated histone H3, myeloperoxidase, neutrophil elastase) and extracellular DNA was observed, which decreased after treatment with DNase or DNase-RM@ (PDA / IDB).

[0026] The protective effect of DNase-RM@(PDA / IDB) on neurons was investigated using the SH-SY5Y cell OGD / R model, and cell viability was assessed using a CCK-8 assay kit. Figure 6 As shown, cell viability decreased after OGD / R treatment, but recovered somewhat after treatment with nanoparticles in each group.

[0027] Thirty minutes after reperfusion, tMCAO model mice were injected via tail vein with an isotonic nanoparticle solution. Twenty-four hours later, the brain was harvested after cardiac perfusion; frozen sections were cut into 1 mm sections for TTC staining to assess infarct area. Figure 7As shown: The model mice given saline had approximately 40% infarction in their brains. After treatment with nanoparticles in each group, the infarct area was reduced. The DNase-RM@(PDA / IDB) group showed the best effect, reducing the infarct area to approximately 5%.

[0028] Experimental Example 1 The method for preparing near-infrared fluorescent probe-labeled nanoparticles involves pre-incubating red blood cell membranes and DiD probes together at room temperature for 1 hour, and then preparing nanoparticles according to the steps in Example 1.

[0029] Thirty minutes after reperfusion, tMCAO model mice were injected via tail vein with a near-infrared fluorescent probe-labeled nanoparticle solution adjusted to isotonicity. Twenty-four hours later, the heart was perfused, and major organs and the brain were harvested for fluorescence imaging. The brain was frozen and sectioned into 1 mm pieces for fluorescence imaging and TTC staining to examine the in vivo and brain distribution behavior of the nanoparticles. Figure 4 As shown, the strongest fluorescence signal was observed in the brains of model mice given DNase-RM@(PDA / IDB), and it co-localized with the infarct region indicated by TTC.

Claims

1. A method for preparing multifunctional biomimetic nanoparticles with an enzyme as a target functional motif, characterized in that, The specific steps are as follows: (1) Dopamine hydrochloride was subjected to oxidative polymerization at room temperature to obtain a polydopamine nanoparticle solution; (2) Stir the polydopamine nanoparticles and the drug described in step (1) at room temperature to obtain a drug-loaded nanoparticle solution; (3) Mix the drug-loaded nanoparticles described in step (2) with mouse erythrocyte membranes and then extrude or sonicate to obtain a coated nanoparticle solution; (4) The coated nanoparticles described in step (3) are co-incubated with DSPE-PEG-AAPVK-biotin at room temperature to obtain a solution of coated nanoparticles with biotin exposed on the surface by responsive peptides. (5) Deoxyribonuclease I was reacted with biotin succinimide ester at room temperature to obtain a biotin-modified enzyme; (6) First, streptavidin was mixed with biotin-modified deoxyribonuclease I and stirred at room temperature. Then, it was added to the nanoparticle solution obtained in step (4) and allowed to stand at room temperature to obtain multifunctional biomimetic nanoparticles with deoxyribonuclease I as the target functional motif.

2. The preparation method according to claim 1, characterized in that, In step (1), pure water, anhydrous ethanol, and ammonia are mixed at room temperature and stirred for 30 minutes. Then, an aqueous solution of dopamine hydrochloride is added, and the mixture is stirred in an open container for 24 hours. The organic solvent and unpolymerized raw materials are removed by ultrafiltration to obtain a polydopamine nanoparticle solution. The volume ratio of pure water, anhydrous ethanol, and ammonia is 1:(0.4~0.6):(0.08~0.1), the mass concentration of ammonia is 25%~28%, the concentration of the aqueous solution of dopamine hydrochloride is 50 mg / mL, and the mass-volume ratio of dopamine hydrochloride to pure water is (4~6):1 mg / mL.

3. The preparation method according to claim 1, characterized in that, In step (2), the drug is first dissolved in an organic solvent and then added to a polydopamine nanoparticle solution and stirred at room temperature for 12 hours; the mass ratio of the drug to the polydopamine nanoparticles is 1:(4~10); the drug is a hydrophobic drug.

4. The preparation method according to claim 3, characterized in that, The drug is idebenone; the organic solvent is dimethyl sulfoxide.

5. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of mouse erythrocyte membrane to drug-loaded nanoparticles is 1:(1~2); the ultrasonic temperature is 4℃ and the ultrasonic time is 2 minutes.

6. The preparation method according to claim 1, characterized in that, In step (4), the coated nanoparticles obtained in step (3) are mixed with DSPE-PEG-AAPVK-biotin and incubated together at room temperature for 1 hour; the mass ratio of the coated nanoparticles obtained in step (3) to DSPE-PEG-AAPVK-biotin is 1: (0.002~0.005).

7. The preparation method according to claim 1, characterized in that, In step (5), firstly, deoxyribonuclease I is dissolved in phosphate buffer, biotin succinimide ester is dissolved in dimethyl sulfoxide, the biotin succinimide ester solution is added to the enzyme solution, and the reaction is stirred at room temperature for 1 hour; the molar ratio of deoxyribonuclease I to biotin succinimide ester is 1: (0.8~1.2).

8. The preparation method according to claim 1, characterized in that, In step (6), the stirring reaction time is 1 hour and the standing time is 1 hour; the molar ratio of streptavidin, biotin coupled with the membrane nanoparticles in step (4), and enzyme modified with biotin in step (5) is 1:(1~3):(1~3).

9. Multifunctional biomimetic nanoparticles obtained by the preparation method according to any one of claims 1-8.

10. The application of the multifunctional biomimetic nanoparticles as described in claim 9 in the preparation of drugs for targeted delivery by multi-target regulation of ischemia-reperfusion injury and neutrophil extracellular traps.

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