Anti-oxidation quercetin nano-particles targeting craniocerebral trauma area as well as preparation method and application of anti-oxidation quercetin nano-particles
By preparing antioxidant quercetin nanoparticles that target the traumatic brain injury area, the problem of insufficient targeting of quercetin in the treatment of traumatic brain injury was solved, and the efficient antioxidant and anti-inflammatory effects of quercetin in the brain injury area were achieved, thus improving the treatment effect of traumatic brain injury.
Patent Information
- Application Number
- CN202511950641.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-02-13
AI Technical Summary
Quercetin has problems in the treatment of traumatic brain injury, such as low solubility, low absorption rate, chemical instability, easy degradation, lack of targeting and difficulty in penetrating the blood-brain barrier. As a result, its bioavailability in the treatment of traumatic brain injury is low and it cannot effectively target the traumatic brain injury area.
Antioxidant quercetin nanoparticles targeting the traumatic brain injury area were prepared by mixing DSPE-PEG2000-CAQK, polypropylene sulfur, lecithin and quercetin in a specific ratio using an organic solvent method. Polypropylene sulfur responded to and consumed reactive oxygen species in an active oxygen environment, and the nanoparticles disintegrated in the brain injury area to release quercetin, thereby achieving antioxidant and anti-inflammatory effects.
It increased the concentration of quercetin at the site of craniocerebral trauma, achieving targeted and controlled drug release, breaking the vicious cycle of reactive oxygen species and neuroinflammation, improving the pathological microenvironment of craniocerebral trauma, and enhancing the therapeutic effect.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of biological medicine, and relates to a drug for treating craniocerebral trauma, in particular to an antioxidant quercetin nanoparticle targeting a craniocerebral trauma region and a preparation method and application thereof BACKGROUND
[0002] Craniocerebral trauma is one of the common clinical neurological diseases, and has become an important challenge in the field of global public health. Epidemiological data shows that the direct and indirect economic loss caused by craniocerebral trauma is more than 400 billion US dollars per year, which constitutes a heavy burden on the social and economic system of each country. From the perspective of clinical outcome, most patients with mild to moderate craniocerebral trauma can fully recover within a few weeks, but about 20-30 % of cases may develop chronic symptoms, such as persistent anxiety, cognitive dysfunction and other neuropsychiatric abnormalities. In contrast, more than 60 % of patients with severe craniocerebral trauma will leave permanent neurological deficits, involving motor coordination disorders, executive function decline and behavioral abnormalities, and the rehabilitation period is significantly prolonged and the prognosis is highly uncertain. From the pathological mechanism level, TBI can be divided into two stages: primary brain injury and secondary brain injury. The former is caused by the direct action of mechanical external force on brain tissue, which is characterized by immediate structural damage and is usually irreversible. The latter develops gradually within a few hours to several days after primary injury, involving complex pathophysiological cascade reactions such as oxidative stress, neuroinflammation and apoptosis, and eventually leads to secondary neurodegeneration. Given the intervention characteristics of secondary injury, the core of modern craniocerebral trauma treatment strategy is to regulate the pathological process related to secondary injury in order to maximize the incidence of long-term neurological deficits.
[0003] The brain, as an organ highly dependent on oxygen supply, is one of the most oxygen-consuming organs in the human body. Although it accounts for only about 2 % of body weight, it consumes about 20 % of oxygen, and its normal function depends on adequate blood supply. Studies have shown that cerebral blood flow accounts for about 15-20 % of cardiac output, and this high perfusion state ensures the high metabolic demand of brain tissue. However, after craniocerebral trauma, ischemia-reperfusion injury leads to interruption of oxygen supply to brain tissue, and thus triggers oxidative stress, which is considered to be a key driving factor of secondary brain injury. In addition, the destruction of the integrity of the blood-brain barrier after craniocerebral trauma allows peripheral harmful substances to penetrate into the brain parenchyma, further exacerbating the pathological process of oxygen supply interruption and oxidative stress. The synergistic effect of insufficient oxygen supply and oxidative stress leads to the generation of a large amount of active oxygen, which causes extensive damage to cell membrane lipids, protein structures and DNA through the initiation of free radical chain reactions, and ultimately leads to cell dysfunction and structural damage. It is worth noting that the endogenous antioxidant system in brain tissue after craniocerebral trauma is dysfunctional, leading to a significant decrease in active oxygen clearance capacity, further exacerbating oxidative stress injury.
[0004] Oxidative stress and neuroinflammation are closely related in the pathological process after traumatic brain injury, forming a vicious cycle of "active oxygen-neuroinflammation". Specifically, active oxygen activates transcription factors such as nuclear factor kappa B, promotes the expression of pro-inflammatory genes, and then induces the release of a large number of inflammatory mediators, such as tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6. At the same time, activated microglia and infiltrating peripheral immune cells during neuroinflammation further produce a large amount of active oxygen, forming a positive feedback loop, continuously aggravating nerve damage. Therefore, breaking the vicious cycle of "active oxygen-neuroinflammation" is one of the key strategies for the treatment of traumatic brain injury. By targeting the regulation of oxidative stress and inhibiting neuroinflammation, it may effectively reduce secondary brain injury, thereby improving patient prognosis.
[0005] Quercetin has shown potential as a therapeutic agent for traumatic brain injury due to its significant antioxidant and anti-inflammatory properties, and its value in the treatment of traumatic brain injury has received widespread attention in recent years. However, quercetin faces multiple limitations in clinical application for traumatic brain injury, specifically as follows:
[0006] ① Low solubility and absorption rate;
[0007] ② Chemically unstable and easily degradable, its strong reducing property is easily oxidized to reduce activity;
[0008] ③ Lack of targeting in drug distribution in vivo
[0009] ④ Quercetin is difficult to effectively penetrate the blood-brain barrier. SUMMARY
[0010] In view of the above technical problems in the prior art, the present application provides an antioxidant quercetin nanoparticle targeting the traumatic brain injury area and its preparation method and application. The nanoparticle for treating traumatic brain injury and its preparation method and application solve the technical problems in the prior art that quercetin applied to the treatment of traumatic brain injury lacks targeting, has low bioavailability, and cannot pass through the blood-brain barrier.
[0011] The present application provides an antioxidant quercetin nanoparticle targeting the traumatic brain injury area, which is prepared from raw materials in the following mass ratio:
[0012] DSPE-PEG 2000 -CAQK 0.12~0.15;
[0013] Polypropylene sulfide 1~1.5;
[0014] Lecithin 0.1~0.2;
[0015] Quercetin 0.12~0.15.
[0016] Further, the antioxidant quercetin nanoparticles targeting the brain trauma area are prepared from raw materials in the following mass ratio:
[0017] DSPE-PEG 2000 -CAQK 0.12;
[0018] Polypropylene sulfide 1;
[0019] Lecithin 0.1;
[0020] Quercetin 0.12.
[0021] The application also provides a preparation method of the antioxidant quercetin nanoparticles targeting the brain trauma area, comprising the following steps:
[0022] 1) DSPE-PEG 2000 -CAQK, polypropylene sulfide, lecithin, and quercetin are weighed according to the mass ratio;
[0023] 2) The above substances are mixed and dissolved in dimethyl sulfoxide solvent, and are shaken in a vortex mixer to fully dissolve the components;
[0024] 3) A centrifuge tube containing a phosphate buffer solution is placed in a heat collecting constant temperature heating magnetic stirrer, the mixture of step 2) is added in a stirring state, and then maintained for 1-3 hours in a stirring state to form antioxidant quercetin nanoparticles.
[0025] Further, the organic solvent is dimethyl sulfoxide.
[0026] The application also provides the use of the antioxidant quercetin nanoparticles in the preparation of a drug for treating brain trauma.
[0027] The antioxidant quercetin nanoparticles targeting the brain trauma area of the application are prepared from DSPE-PEG 2000 -CAQK, polypropylene sulfide, lecithin, and quercetin are fully dissolved in dimethyl sulfoxide, and are dropped into a phosphate buffer solution to prepare by an organic solvent injection method.
[0028] The quercetin is a natural antioxidant and anti-inflammatory flavonoid, which can realize sustained and effective elimination of active oxygen and play an anti-inflammatory role, but has low bioavailability, is difficult to pass through the blood-brain barrier, and lacks targeting in systemic application.
[0029] The DSPE-PEG 2000 -CAQK is a lipid molecule constructed based on a cystine-alanine-glutamic acid-lysine short peptide, which can be combined with chondroitin sulfate proteoglycans specifically exposed or expressed after injury, so as to endow the nanoparticles with the ability to target the brain trauma area, and the structural formula is as follows:
[0030] .
[0031] The polythia-propylene in the polythia-propylene can respond to and consume active oxygen in an active oxygen environment, and its structural formula is as follows:
[0032] .
[0033] Lecithin mainly plays a role in structural stabilization and interface regulation in nanoparticles. Its amphiphilic structure can self-assemble into a stable lipid layer in an aqueous phase, coating the core of the nanoparticles, improving the encapsulation efficiency of hydrophobic drugs and reducing aggregation. At the same time, as a natural component of the cell membrane, lecithin can significantly improve the biocompatibility and blood stability of nanoparticles, reduce non-specific protein adsorption and in vivo clearance, and is beneficial to improve the in vivo delivery efficiency, especially suitable for the nervous system related nanometer delivery system.
[0034] In order to overcome the clinical difficulties of the conventional application mode of quercetin, and at the same time reduce the accumulation of active oxygen in the microenvironment after trauma, the application also provides a preparation method of the above-mentioned antioxidant quercetin nanoparticles targeting the craniocerebral trauma area. The designed and prepared quercetin nanoparticles can target the brain injury area, respond to and consume active oxygen while releasing quercetin, break the vicious cycle of "active oxygen-neuroinflammation", and treat craniocerebral trauma.
[0035] The specific treatment steps of the application are as follows: ① Intravenous administration of quercetin nanoparticles after craniocerebral trauma; ② Quercetin nanoparticles can be targeted to accumulate in the brain injury area; ③ Quercetin nanoparticles reaching the injury area can respond to and consume active oxygen in the injury area due to the presence of polythia-propylene, and cause the disintegration of the nanoparticles, release quercetin, and play an anti-inflammatory role. Quercetin nanoparticles overcome the clinical difficulties of conventional drug preparations, improve the concentration of drugs in the brain trauma area, and achieve controlled release of drugs, timely and effectively improve the effect of craniocerebral trauma treatment, and improve the level of craniocerebral trauma treatment.
[0036] Compared with the prior art, the technical effect of the present application is positive and obvious. In order to overcome the problems in clinical application of quercetin, such as insufficient targeting, low local effective concentration in craniocerebral trauma and unstable treatment effect in conventional administration mode, and effectively regulate the abnormal accumulation of reactive oxygen species in the microenvironment after craniocerebral trauma, the present application provides an antioxidant quercetin nanoparticle which can target the craniocerebral trauma area by intravenous administration and a treatment method thereof. The designed and prepared quercetin nanoparticle can specifically enrich in the brain injury area after circulating in the body, and rely on the response characteristics of the polypropylene sulfide structure introduced into the inside to selectively consume reactive oxygen species in the injury area, thereby inducing the disintegration of the nanoparticle and realizing the in-situ release of quercetin, so as to simultaneously play the roles of antioxidant and anti-inflammatory in the local area, block the vicious cycle of "reactive oxygen species-neuroinflammation", and achieve the purpose of improving the pathological microenvironment of craniocerebral trauma and improving the treatment effect. BRIEF DESCRIPTION OF DRAWINGS
[0037] Figure 1 The structure of the quercetin nanoparticle prepared in the present application (bar = 200 nm).
[0038] Figure 2 The particle size and potential of the quercetin nanoparticle prepared in the present application.
[0039] Figure 3 The ability of the quercetin nanoparticle prepared in the present application to target the brain injury area (bar = 100 μm).
[0040] Figure 4 The ability of the quercetin nanoparticle prepared in the present application to consume reactive oxygen species (bar = 200 μm).
[0041] Figure 5 The quercetin nanoparticle prepared in the present application has the effect of protecting the blood-brain barrier.
[0042] Figure 6 The quercetin nanoparticle prepared in the present application can reduce tissue damage (bar = 200 μm).
[0043] Figure 7 The quercetin nanoparticle prepared in the present application has the effect of protecting neurons (bar = 200 μm).
[0044] Figure 8 The quercetin nanoparticle prepared in the present application has the effect of improving neuroinflammation (bar = 100 μm).
[0045] Figure 9 The quercetin nanoparticle prepared in the present application has the effect of improving the neurological function after craniocerebral trauma.
[0046] Figure 10 The quercetin nanoparticles prepared by the present application have good biocompatibility (bar = 100 μm). DETAILED DESCRIPTION
[0047] The present application is further illustrated below with examples.
[0048] Example 1: Preparation and characterization of quercetin nanoparticles
[0049] The preparation of quercetin nanoparticles is as follows:
[0050] DSPE-PEG 2000 -CAQK (from Xi'an Ruixi Biological Technology Co., Ltd.) 0.12 mg, polypropylene sulfide (from Xi'an Ruixi Biological Technology Co., Ltd.) 0.1 mg, lecithin (from MedChemExpress) 0.1 mg, quercetin 0.12 mg (from MedChemExpress).
[0051] After mixing the above substances, dissolve them in 200 μL dimethyl sulfoxide, shake in a vortex mixer to fully dissolve each component, place a centrifuge tube containing phosphate buffer solution (800 μL) in a heat collecting constant temperature heating magnetic stirrer, slowly drop the above mixture under high speed stirring at 25°C, and then maintain slow stirring at 25°C for about 2 hours to form quercetin nanoparticles (CP / Q).
[0052] As Figure 1 The structure of quercetin nanoparticles with different components is shown.
[0053] As Figure 2 The particle size and potential of quercetin nanoparticles with different components are shown.
[0054] CP is a nanoparticle without quercetin, i.e. DSPE-PEG 2000 -CAQK (from Xi'an Ruixi Biological Technology Co., Ltd.) 0.12 mg, polypropylene sulfide (from Xi'an Ruixi Biological Technology Co., Ltd.) 0.1 mg, lecithin (from MedChemExpress) 0.1 mg. After mixing the above substances, dissolve them in 200 μL dimethyl sulfoxide, shake in a vortex mixer to fully dissolve each component, place a centrifuge tube containing phosphate buffer solution (800 μL) in a heat collecting constant temperature heating magnetic stirrer, slowly drop the above mixture under high speed stirring at 25°C, and then maintain slow stirring at 25°C for about 2 hours to form nanoparticles (CP)
[0055] CPLGA / Q is a nanoparticle with a hydrophobic core composed of polylactic acid-glycolic acid copolymer, i.e. DSPE-PEG2000 -CAQK (from Xi'an Ruixi Biological Technology Co., Ltd.) 0.12 mg, polylactic acid-glycolic acid copolymer (from MedChemExpress) 0.1 mg, lecithin (from MedChemExpress) 0.1 mg. After mixing the above substances, dissolve them in 200 μL dimethyl sulfoxide, shake in a vortex mixer to fully dissolve each component, place a centrifuge tube containing a phosphate buffer solution (800 μL) in a heat constant temperature magnetic stirrer, slowly drop the above mixture into the centrifuge tube under the condition of high speed stirring at 25 °C, and then maintain it under the condition of slow stirring at 25 °C for about 2 hours to form nanoparticles (CPLGA / Q)
[0056] Example 2: Targeting of quercetin nanoparticles
[0057] Take an appropriate amount of quercetin nanoparticles of Example 1 to perform a nanoparticle targeting experiment on a mouse model of craniocerebral trauma.
[0058] Inject PBS and DiR fluorescently labeled CP / Q into the mouse body, and observe the drug enrichment in the damaged area of the brain tissue section after injection. As shown in Figure 3 , the quercetin nanoparticles prepared by the present application can be effectively targeted and enriched in the craniocerebral injury area.
[0059] Example 3: Ability of quercetin nanoparticles to consume reactive oxygen species
[0060] Take an appropriate amount of quercetin nanoparticles of Example 1, co-culture with reactive oxygen species-damaged neuron cells, and observe the ability of the nanoparticles to consume reactive oxygen species, as shown in Figure 4 (Control: untreated group after injury; CP / Q: treatment group given quercetin nanoparticles after injury).
[0061] Use the reactive oxygen species detection kit to detect the intracellular ROS level. Seed the cells in a 96-well plate at a density of 1 × 10 5 Incubate the cells with 10 μL nanoparticles and hydrogen peroxide (0.5 mM or 0.25 mM) for 2 hours. The control group is not treated with anything. Then, use 10 μM dichlorofluorescin diacetate (2',7'-Dichlorofluorescin Diacetate, DCFH-DA, from MedChemExpress) in serum-free medium at 37 °C for 30 min. After removing the DCFH-DA, observe the generation of intracellular ROS using a fluorescence microscope (excitation 488 nm, emission wavelength 525 nm). As shown in Figure 4 , the strength of the reactive oxygen species fluorescent probe indicates that quercetin nanoparticles can effectively consume the reactive oxygen species of neuron cells.
[0062] Example 4: Ability of quercetin nanoparticles to protect the blood brain barrier
[0063] Example 4: Ability of quercetin nanoparticles to protect the blood brain barrier Figure 5 (Control: untreated group of craniocerebral trauma; CP / Q: treatment group of craniocerebral trauma with quercetin nanoparticles).
[0064] Each group of mice was intravenously injected with 2% Evans Blue (from MedChemExpress); 3h after injection, the dye was allowed to distribute; the mice were then heart perfused with saline to remove the non-bound dye from the blood vessels; the brain tissue was removed, the brain surface was rinsed with saline and photographed; as shown in Figure 5 Example 4: Ability of quercetin nanoparticles to protect the blood brain barrier
[0065] Example 5: Ability of quercetin nanoparticles to reduce tissue damage
[0066] Example 5: Ability of quercetin nanoparticles to reduce tissue damage Figure 6 (Control: untreated group of craniocerebral trauma; CP / Q: treatment group of craniocerebral trauma with quercetin nanoparticles).
[0067] The brain tissue of each group of mice was subjected to histological analysis. The brain tissue was fixed with 10% formalin, paraffin-embedded and then cut into 20 pm sections using a microtome. These sections were then subjected to hematoxylin-eosin staining and observed under a microscope.
[0068] Example 5: Ability of quercetin nanoparticles to reduce tissue damage Figure 6 Example 5: Ability of quercetin nanoparticles to reduce tissue damage
[0069] Example 6: Ability of quercetin nanoparticles to protect neurons
[0070] Example 6: Ability of quercetin nanoparticles to protect neurons Figure 7 (Control: untreated group of craniocerebral trauma; CP / Q: treatment group of craniocerebral trauma with quercetin nanoparticles).
[0071] The brain tissue of each group of mice was subjected to histological analysis. The brain tissue was fixed with 10% formalin, paraffin-embedded and then cut into 20 pm sections using a microtome. These sections were then subjected to hematoxylin-eosin staining and observed under a microscope.
[0072] Example 6: Ability of quercetin nanoparticles to protect neurons Figure 7 Example 6: Ability of quercetin nanoparticles to protect neurons
[0073] Example 7: Effect of quercetin nanoparticles on improving neuroinflammation
[0074] The effect of quercetin nanoparticles on improving neuroinflammation was detected by immunofluorescence staining, as shown in Figure 8 (Control: untreated group of craniocerebral trauma; CP / Q: treatment group of craniocerebral trauma given quercetin nanoparticles).
[0075] The brain tissue of each group of mice was taken for histological analysis. The brain tissue was fixed with 10% formalin, paraffin-embedded, and then cut into 20 μm sections with a paraffin microtome. These sections were then subjected to immunofluorescence staining and observed under a microscope.
[0076] As shown in Figure 8 , the quercetin nanoparticles prepared in Example 1 of the present application can improve neuroinflammation after craniocerebral trauma (bar = 200 μm).
[0077] Example 8: Effect of quercetin nanoparticles on improving neurological function
[0078] The Morris water maze was used to evaluate the improvement of learning and memory ability after craniocerebral trauma by quercetin nanoparticles, as shown in Figure 10 (Control: untreated group of craniocerebral trauma; CP / Q: treatment group of craniocerebral trauma given quercetin nanoparticles).
[0079] The brain tissue of each group of mice was taken for the Morris water maze experiment, and the movement trajectory of the mice during the learning and memory stage was recorded.
[0080] As shown in Figure 9 , the quercetin nanoparticles prepared in Example 1 of the present application have an effect of improving neurological function.
[0081] Example 9: Biocompatibility of quercetin nanoparticles
[0082] Hematoxylin-eosin staining was used to evaluate the effect of quercetin nanoparticles on other organs, as shown in Figure 10 .
[0083] The heart, liver, spleen, lung, and kidney of each group of mice were taken for histological analysis. The brain tissue was fixed with 10% formalin, paraffin-embedded, and then cut into 20 μm sections with a paraffin microtome. These sections were then subjected to hematoxylin-eosin staining and observed under a microscope.
[0084] As shown in Figure 10 , the quercetin nanoparticles prepared in Example 1 of the present application have good biocompatibility.
Claims
1. An antioxidant quercetin nanoparticle targeting the traumatic brain injury region, characterized in that, It is prepared from the following raw materials in the following mass ratio: DSPE-PEG 2000 -CAQK 0.12~0.15; Polypropylene sulfur 1~1.5; Lecithin 0.1~0.2; Quercetin 0.12~0.
15.
2. The antioxidant quercetin nanoparticles targeting the traumatic brain injury region according to claim 1, characterized in that, It is prepared from the following raw materials in the following mass ratio: DSPE-PEG 2000 -CAQK 0.12; Polypropylene sulfur 1; Lecithin 0.1; Quercetin 0.
12.
3. The method for preparing antioxidant quercetin nanoparticles targeting the traumatic brain injury region as described in claim 1 or 2, characterized in that... Includes the following steps: 1) Weigh out DSPE-PEG according to the mass ratio. 2000 -CAQK, polypropylene sulfur, lecithin, quercetin; 2) After mixing the above substances, dissolve them completely in an organic solvent; 3) Place the container containing the phosphate buffer solution in a heat-collecting constant-temperature magnetic stirrer, add the mixture from step 2) above while stirring, and then maintain stirring for 1 to 3 hours to form antioxidant quercetin nanoparticles.
4. The method for preparing antioxidant quercetin nanoparticles targeting the traumatic brain region according to claim 3, characterized in that: The organic solvent is dimethyl sulfoxide.
5. Use of the antioxidant quercetin nanoparticles according to claim 1 or 2 in the preparation of a medicament for treating craniocerebral trauma.