Quercetin composite nanoparticle, and preparation method and application thereof
Through the preparation of quercetin composite nanoparticles, the low bioavailability and administration problems caused by the hydrophobicity of quercetin were solved, the efficient intravenous administration of quercetin and the inhibition of oxidative stress were achieved, and a new treatment method for ischemia-reperfusion injury was provided.
Patent Information
- Application Number
- CN202510688272.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-27
- Publication Date
- 2025-09-19
AI Technical Summary
The existing technology lacks effective drugs for treating ischemia-reperfusion injury, especially because the hydrophobicity of quercetin limits its bioavailability and administration mode, resulting in insufficient treatment convenience.
By preparing quercetin composite nanoparticles, methylcobalamin and quercetin are self-assembled to form a stable aqueous dispersion, which can be administered intravenously, improve the dispersibility and stability of quercetin, and exert the effects of inhibiting oxidative stress and inflammatory factors.
The efficient intravenous administration of quercetin was achieved, the potential toxic side effects were reduced, a new treatment strategy for ischemia-reperfusion injury was provided, and the treatment effect was significantly improved.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of nanopharmaceuticals, and in particular relates to a quercetin composite nanoparticle, a preparation method thereof and an application thereof. Background Art
[0002] Ischemia-reperfusion injury (IRI) is a condition characterized by exacerbated cellular damage and inflammation when blood supply is restored to tissues following ischemia. Oxidative stress is a core mechanism in this process, and its effects on organs are widespread and severe. Currently, there are no effective treatments for this condition.
[0003] It is known that a variety of causes can lead to ischemia-reperfusion injury, such as cerebral infarction and cardiac arrest, so it is very necessary to develop effective intervention strategies. On the one hand, there is currently a lack of corresponding drugs. On the other hand, the way the drug is administered also affects the convenience of treatment. For example, after a cerebral infarction patient falls into a coma and is unable to follow the doctor's orders to take the medication on their own, intravenous injection becomes an effective way of administration. Therefore, convenient drug administration for ischemia-reperfusion injury is also a necessary task in the development of such drugs.
[0004] Quercetin has potential therapeutic effects on ischemia-reperfusion injury. However, its hydrophobicity significantly limits its clinical application. Therefore, it is necessary to develop a quercetin composite nanoparticle technology. Summary of the Invention
[0005] To address the deficiencies of the existing technology, the present invention provides a quercetin composite nanoparticle, a preparation method, and its application. This invention addresses the problem that quercetin's hydrophobic properties limit its bioavailability and administration. By preparing stably dispersed quercetin composite nanoparticles, the present invention overcomes the toxic and side effects of traditional solubilization techniques (such as organic solvents and surfactants), providing a new path for the development of quercetin as a clinical drug candidate.
[0006] The technical solutions provided by the present invention are as follows:
[0007] A quercetin composite nanoparticle comprises quercetin and an amphiphilic molecule, wherein the quercetin and the amphiphilic molecule are combined through intermolecular forces.
[0008] In the above technical solution:
[0009] In the treatment of ischemia-reperfusion injury, quercetin can inhibit oxidative stress and inflammatory factors;
[0010] On the one hand, methylcobalamin combines with quercetin through intermolecular forces to achieve self-assembly, significantly improving the dispersibility and stability of quercetin, so that it can be used to prepare aqueous dispersion preparations, such as injections; on the other hand, in the treatment of ischemia-reperfusion injury, methylcobalamin plays a role in nourishing nerves, assisting quercetin's oxidative stress inhibition and inflammatory factor inhibition, thereby achieving the treatment of ischemia-reperfusion injury.
[0011] Specifically, the weight percentage of quercetin in the quercetin composite nanoparticles is 60-80%, preferably 70%.
[0012] Specifically, the quercetin is anhydrous quercetin.
[0013] Specifically, the amphiphilic molecules include but are not limited to methylcobalamin, and other pharmaceutically acceptable amphiphilic molecules that have the same effect as quercetin.
[0014] Specifically, the particle size of the quercetin composite nanoparticles is 198-288 nm.
[0015] The present invention also provides a method for preparing quercetin composite nanoparticles, comprising the following steps:
[0016] 1) dissolving quercetin in a solvent to obtain solution I; dissolving the amphiphilic molecule in the same solvent to obtain solution II; mixing solution I and solution II and then sonicating to obtain solution III;
[0017] 2) adding the solution III dropwise into an aqueous acid solution to form particles;
[0018] 3) The dispersion obtained in step 2) is dialyzed, ultrafiltered, resuspended, and freeze-dried to obtain quercetin composite nanoparticles.
[0019] The nanoparticles prepared by the above technical solution are spherical, have uniform particle size, a drug loading capacity of about 70%, good dispersibility, do not require a carrier, and have small differences between batches.
[0020] Specifically, in step 1):
[0021] The solvent includes but is not limited to dimethyl sulfoxide;
[0022] The concentration of the solution I is 1 mg / mL-10 mg / mL, preferably 3 mg / mL-6 mg / mL, more preferably 4 mg / mL;
[0023] The concentration of the solution II is 1 mg / mL-10 mg / mL;
[0024] The mass ratio of the solution I to the solution II is (1:10)-(10:10).
[0025] Specifically, in step 2):
[0026] The pH of the aqueous solution of the acid is 6-6.4, preferably, the pH is 6.0; the acid includes but is not limited to hydrochloric acid;
[0027] The volume ratio of the solution III to the acid aqueous solution is 1:(10-15), preferably 75:1000.
[0028] Preferably, the solution III is added dropwise to the aqueous acid solution under vortex conditions, and the prepared quercetin composite nanoparticles are well dispersed in water and have uniform and stable particle sizes.
[0029] Specifically, in step 3):
[0030] The pore size of the dialysis membrane is 1000-10000Da, preferably 3500Da;
[0031] The pore size of the ultrafiltration membrane is 1000-50000Da, preferably 10000Da;
[0032] The resuspension is of the same type as the acid aqueous solution in step 2), and preferably has a pH of 6.0.
[0033] The present invention also provides an application of the quercetin composite nanoparticles for preparing a medicine for treating ischemia-reperfusion injury.
[0034] Specifically, the ischemia-reperfusion injury disease includes cerebral ischemia-reperfusion injury caused by cerebral infarction.
[0035] Preferably, it is used as an oxidative stress inhibitor to prepare a drug for treating ischemia-reperfusion injury.
[0036] Preferably, it is used as an inflammatory factor inhibitor to prepare a drug for treating ischemia-reperfusion injury.
[0037] Preferably, the drug is an intravenous injection.
[0038] Specifically, the dosage of quercetin composite nanoparticles is 4-6 mg / kg per day, preferably 5 mg / kg per day.
[0039] The beneficial effects of the present invention are as follows:
[0040] 1) No need to introduce external carrier components, reducing potential toxic side effects;
[0041] 2) Based on the dispersibility of quercetin composite nanoparticles, intravenous administration can be achieved;
[0042] 3) Provide new treatment strategies for organ ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS
[0043] Figure 1 This is a particle size distribution diagram of Example 1.
[0044] Figure 2 This is a TEM image of Example 1.
[0045] Figure 3 This is a particle size distribution diagram of Example 2.
[0046] Figure 4 This is another particle size distribution diagram of Example 2.
[0047] Figure 5 This is a graph of particle size stability of Example 3.
[0048] Figure 6 This is a potential stability diagram of Example 3.
[0049] Figure 7 This is a graph showing drug loading data for different batches in Example 4.
[0050] Figure 8 This is a diagram showing the cell biosafety of Example 5.
[0051] Figure 9 This is a diagram of the glucose-oxygen deprivation / reperfusion cell model of Example 6.
[0052] Figure 10 This is the in vivo biosafety diagram of Example 7.
[0053] Figure 11 This is a diagram showing the effect of Example 8 in the cerebral ischemia-reperfusion injury model. DETAILED DESCRIPTION
[0054] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.
[0055] Unless otherwise specified, the test methods used in the examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.
[0056] Quercetin was anhydrous quercetin, and all other reagents used were of analytical grade.
[0057] The aqueous solution of the acid is dilute hydrochloric acid with a pH of 6.0.
[0058] The pore size of the dialysis membrane is 3500 Da.
[0059] The pore size of the ultrafiltration membrane is 10,000 Da.
[0060] Example 1
[0061] Quercetin and methylcobalamin self-assemble to prepare quercetin composite nanoparticles, the steps are as follows:
[0062] 1) Weigh quercetin and dissolve it in dimethyl sulfoxide to a concentration of 4 mg / mL;
[0063] 2) Weigh methylcobalamin and dissolve it in dimethyl sulfoxide to a concentration of 10 mg / mL;
[0064] 3) Take 25 μL of the solution from step 1) and 50 μL of the solution from step 2), mix them, and sonicate for 2 minutes to a total volume of 75 μL;
[0065] 4) adding the solution obtained in step 3) dropwise to a pH 6.0 aqueous solution while vortexing, wherein the amount of the solution obtained in step 3) added is 75 μL, which is added to 1000 μL of the pH 6.0 aqueous solution in five equal portions;
[0066] 5) The dispersion obtained in step 4) is dialyzed against a pH 6.0 solution, and finally resuspended, quickly frozen, and then lyophilized in a lyophilizer to obtain the nanomedicine.
[0067] The results are as follows Figure 1-2 As shown, the self-assembled quercetin composite nanoparticles can be well dispersed in aqueous solution and have uniform particle size distribution.
[0068] Example 2
[0069] Preparation of quercetin composite nanoparticles and demonstration of the effect of different quercetin concentrations on nanoparticle synthesis were performed as follows:
[0070] 1) Weigh quercetin and dissolve it in dimethyl sulfoxide to prepare concentrations of 1 mg / mL and 10 mg / mL respectively;
[0071] 2) Weigh methylcobalamin and dissolve it in dimethyl sulfoxide to a concentration of 10 mg / mL;
[0072] 3) Take 25 μL of the solution from step 1) and 50 μL of the solution from step 2), mix them, and sonicate for 2 minutes to a total volume of 75 μL;
[0073] 4) adding the solution obtained in step 3) dropwise to a pH 6.0 aqueous solution while vortexing, wherein the amount of the solution obtained in step 3) added is 75 μL, which is added to 1000 μL of the pH 6.0 aqueous solution in five equal portions;
[0074] 5) The dispersion obtained in step 4) is dialyzed against a pH 6.0 solution, and finally resuspended, quickly frozen, and then freeze-dried in a freeze dryer to obtain the nanomedicine.
[0075] The results are as follows Figure 3-4 As shown, Figure 3 The corresponding quercetin concentration is 1 mg / mL. Figure 4 The corresponding quercetin concentration is 10 mg / mL. It can be seen that the self-assembled nanomedicine can be well dispersed in the aqueous solution with uniform particle size distribution.
[0076] Example 3
[0077] The steps for preparing quercetin composite nanoparticles and demonstrating the stability of the nanomedicine are as follows:
[0078] 1) Weigh quercetin and dissolve it in dimethyl sulfoxide to a concentration of 4 mg / mL;
[0079] 2) Weigh methylcobalamin and dissolve it in dimethyl sulfoxide to a concentration of 10 mg / mL;
[0080] 3) Take 25 μL of the solution from step 1) and 50 μL of the solution from step 2), mix them, and sonicate for 2 minutes to a total volume of 75 μL;
[0081] 4) adding the solution obtained in step 3) dropwise to a pH 6.0 aqueous solution while vortexing, wherein the amount of the solution obtained in step 3) added is 75 μL, which is added to 1000 μL of the pH 6.0 aqueous solution in five equal portions;
[0082] 5) dialyzing the dispersion obtained in step 4) against a pH 6.0 solution, and finally resuspending and quick-freezing the dispersion, followed by freeze drying in a freeze dryer to obtain the nanomedicine;
[0083] 6) The nanomedicine prepared in step 4) was diluted to 0.1 mg / mL by adding a pH 6.0 aqueous solution, and then the particle size and surface potential of the nanomedicine were continuously measured by a dynamic light scattering particle size analyzer (DLS).
[0084] The results are as follows Figure 5-6 As shown in the figure, the quercetin composite nanoparticles prepared by vortexing are well dispersed in water, and the particle size is uniform and stable.
[0085] Example 4
[0086] Drug loading of quercetin and methylcobalamin in nanomedicines
[0087] With reference to the preparation method of Example 1, quercetin at a concentration of 4 mg / mL and methylcobalamin at a concentration of 10 mg / mL were weighed, the volume ratio of quercetin solution to methylcobalamin was 1: 2, and the composite dispersion of quercetin and methylcobalamin was added dropwise to 1000 μL of a pH 6.0 aqueous solution to a total volume of 75 μL. The prepared quercetin composite nanoparticles were freeze-dried and the powder was quantified and dissolved in dimethyl sulfoxide. The content of methylcobalamin was detected by atomic emission spectroscopy to obtain the content of quercetin.
[0088] Repeat the above steps 3 times, the result is as follows Figure 7As shown, the preparation process of the prepared quercetin composite nanoparticles is stable and the drug loading capacity is high.
[0089] Example 5
[0090] Inhibitory effect of quercetin composite nanoparticles on cell proliferation
[0091] 1) preparing quercetin composite nanoparticles into dispersions with different concentration gradients;
[0092] 2) After quercetin composite nanoparticles were co-incubated with various cells for 24 hours, the cell viability was detected using the CCK8 method.
[0093] The results are as follows Figure 8 As shown, the results of 24h co-incubation of neural cells Sy5y and microglial cells BV2 with quercetin composite nanoparticles indicate that quercetin composite nanoparticles have no significant inhibitory effect on cell proliferation.
[0094] Example 6
[0095] Effects of quercetin composite nanoparticles on anti-oxidative stress
[0096] 1) A glucose-oxygen deprivation / reperfusion model was established using BV2 neural cells. Microglial BV2 cells supplemented with conventional culture medium served as the control group. Microglial BV2 cells supplemented with glucose-free culture medium were incubated in a 37°C hypoxic incubator with 1% O₂, 5% CO₂, and 94% N₂ for 9 hours, followed by incubation with conventional culture medium in a standard incubator for 3 hours, serving as the glucose-oxygen deprivation group.
[0097] 2) A 40 μg / mL dispersion of quercetin composite nanoparticles and BV2 neurons was prepared using a sugar-free medium and incubated in a 37°C hypoxic incubator with 1% O2, 5% CO2, and 94% N2 for 9 hours. The cells were then incubated in a conventional incubator with regular medium for 3 hours to serve as the glucose-oxygen deprivation + nanomedicine group.
[0098] 3) CCK8 was used to assess cell viability.
[0099] The results are as follows Figure 9 As shown, quercetin composite nanoparticles can effectively inhibit glucose-oxygen deprivation / reperfusion injury of cells, and the cell proliferation is increased by about 30% compared with the glucose-oxygen deprivation group.
[0100] Example 7
[0101] Evaluation of the biosafety of quercetin composite nanoparticles in vivo
[0102] 1) 5-week-old female C57BL / 6 mice were selected and divided into 2 groups (3 mice in each group);
[0103] 2) In vivo biosafety assessment of quercetin composite nanoparticles injected via tail vein;
[0104] 3) The two groups received PBS (control group) and quercetin composite nanoparticles (5 mg / kg, nanodrug group) treatment, respectively. The main organs were collected for HE staining 24 hours later.
[0105] The results are as follows Figure 10 As shown, no obvious systemic toxicity or side effects were observed after the use of quercetin composite nanoparticles.
[0106] Example 8
[0107] Application of quercetin composite nanoparticles in cerebral ischemia-reperfusion injury model.
[0108] 1) Five-week-old female C57BL / 6 mice were selected and a cerebral ischemia-reperfusion injury model was established using the suture embolization method;
[0109] 2) After successful modeling, the mice were divided into 5 groups, with 5 mice in each group;
[0110] 3) The mice were divided into sham operation group (control group), model group, quercetin group (intraperitoneal injection of quercetin 50 mg / kg), methylcobalamin group (methylcobalamin drug group 1.5 mg / kg) and nanodrug group (quercetin composite nanoparticles 5 mg / kg). The injection was performed 1 hour after ischemia, and the behavior of the mice was observed and recorded for analysis.
[0111] The results are as follows Figure 11 The cerebral ischemic area and behavioral test results of the quercetin composite nanoparticle treatment group were significantly improved compared with those of the other groups.
[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A quercetin composite nanoparticle, characterized in that: include: Quercetin and the amphiphilic molecule are bound to each other through intermolecular forces.
2. The quercetin composite nanoparticles according to claim 1, characterized in that: The weight percentage of quercetin in the quercetin composite nanoparticles is 60-80%; And / or, the quercetin is anhydrous quercetin.
3. The quercetin composite nanoparticles according to claim 1, characterized in that: The amphiphilic molecule includes but is not limited to methylcobalamin.
4. The quercetin composite nanoparticles according to claim 1, wherein: The particle size of the quercetin composite nanoparticles is 198-288 nm.
5. A method for preparing the quercetin composite nanoparticles according to any one of claims 1 to 4, characterized in that: The following steps are involved: 1) dissolving quercetin in a solvent to obtain solution I; dissolving the amphiphilic molecule in the same solvent to obtain solution II; mixing solution I and solution II and then sonicating to obtain solution III; 2) adding the solution III dropwise into an aqueous acid solution to form particles; 3) The dispersion obtained in step 2) is dialyzed, ultrafiltered, resuspended, and freeze-dried to obtain quercetin composite nanoparticles.
6. The method for preparing quercetin composite nanoparticles according to claim 5, wherein: In step 1): The solvent includes but is not limited to dimethyl sulfoxide; The concentration of the solution I is 1 mg / mL-10 mg / mL; The concentration of the solution II is 1 mg / mL-10 mg / mL; The mass ratio of the solution I to the solution II is (1:10)-(10:10).
7. The method for preparing quercetin composite nanoparticles according to claim 5, wherein: In step 2): The pH of the aqueous solution of the acid is 6-6.4; the acid includes but is not limited to hydrochloric acid; The solution III was added dropwise to the aqueous acid solution under vortexing conditions; The volume ratio of the solution III to the aqueous solution of the acid is 1:(10-15).
8. The method for preparing quercetin composite nanoparticles according to claim 5, wherein: In step 3): The pore size of the dialysis membrane is 1000-10000Da; The pore size of ultrafiltration membrane is 1000-50000Da; The resuspension is of the same type as the acid aqueous solution in step 2).
9. A use of the quercetin composite nanoparticles according to any one of claims 1 to 4, characterized in that: Used for preparing medicines for treating ischemia-reperfusion injury.
10. The use according to claim 9, characterized in that: The ischemia-reperfusion injury disease includes cerebral ischemia-reperfusion injury caused by cerebral infarction; or, as an oxidative stress inhibitor to prepare a drug for treating ischemia-reperfusion injury disease; Alternatively, it can be used as an inflammatory factor inhibitor to prepare a drug for treating ischemia-reperfusion injury; Alternatively, the drug is administered intravenously.